All the plant material is from the model organism Arabidopsis thaliana. The marker lines used are: pVHA-a1::VHA-a1-GFP (Dettmer et al., 2006), pCLC2::CLC2-GFP (Konopka et al., 2008), p35S::CLC1-GFP (Wang et al., 2013), p35S::N-ST-YFP (Grebe et al., 2003), pPIN2::PIN2-Dendra eir1-1 (Salanenka et al., 2018), pPIN2::PIN2-GFP x p35S::ARA7-RFP (Ueda et al., 2004; Xu, 2005; Zhang et al., 2016), pUBQ10::CITRINE-1xPH(FAPP1) (Simon et al., 2014), pPEPR1::PEPR1-GFP pepr1 pepr2 (Ortiz-Morea et al., 2016), XVE::AUXILIN-LIKE2 x pPIN2::PIN2-Dendra eir1-1 (Adamowski et al., 2018); pVHA-a1::VHA-a1-RFP x pPIN2::PIN2-GFP eir1-1 was made by crossing pVHA-a1::VHA-a1-RFP (Dettmer et al., 2006) and eir1-1 pPIN2::PIN2-GFP (Xu, 2005).

IAA (indole 3-acetic acid, Duchefa Biochemie, Amsterdam, The Netherlands, I0901.0025) was dissolved in ethanol (EtOH) or DMSO (Dimethylsulfoxid) to a stock concentration of 10 mM (in DMSO), 1 mM (in DMSO), 100 μM (in EtOH), or 10 μM (in EtOH). 1-NAA (Sigma-Aldrich, St Louis, MO, N0640) dissolved in DMSO to a stock concentration of 10 mM. BFA (Brefeldin-A, Sigma-Aldrich, St Louis, MO, B7651) was dissolved in DMSO to a stock concentration of 50 mM. CHX (Sigma-Aldrich, St Louis, MO, C1988) was dissolved in DMSO to a stock concentration of 10 mM. FM4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) Hexatrienyl) Pyridinium Dibromide, Life Technology, T-13320) was dissolved in water to a stock concentration of 2 mM. Blebbistatin (Santa Cruz Biotechnology, San Diego, CA, sc-204253) was dissolved in DMSO to a stock concentration of 1 M. pep1 (peptide sequence: ATKVKAKQRGKEKVSSGRPGQHN (Ortiz-Morea et al., 2016), commercially synthesized by EZbiolab), was dissolved in water to a stock concentration of 200 μM. β-estradiol (Sigma-Aldrich, E8875) was dissolved in DMSO to a stock concentration of 50 mM. For Western blot analysis, the following antibodies were used: primary rabbit anti-PIN2 1:2,000 (produced and processed in lab, Abas et al., 2006), mouse anti-actin 1:5000 (Sigma-Aldrich, St Louis, MO, A0480), mouse anti-His 1:1000 (GE Healthcare, Chicago, IL) and secondary anti-rabbit IgG antibody conjugated to horseradish peroxidase (HRP) 1:10,000 (GE Healthcare, Chicago, IL, NA934). Membranes were developed using the SuperSignal Chemiluminiscence solutions (SuperSignal West Femto, Thermo Scientific, Waltham, MA). For immunolocalization the following primary and secondary antibodies were used: rabbit anti-ARF1 1:500 (Agrisera, Vännäs, Sweden, AS08325), goat anti-PIN1 1:600 (SantaCruz Technologies, San Diego, CA, sc-27163), mouse anti-GFP 1:500 (Sigma, St Louis, MO, G6539) and rabbit anti-PIN2 1:1,000 (produced and processed in lab, Abas et al., 2006), donkey anti-goat antibody coupled to Alexa Fluor 488 1:600 (Thermo Fisher Scientific, Waltham, MA, A11055), goat anti-mouse antibody coupled to Alexa Fluor 594 1:600 (Abcam, Cambridge, MA, 150116) goat anti-rabbit antibody coupled to Alexa Fluor 488 (Invitrogen, Carlsbad, CA, A11034), and sheep anti-rabbit antibody coupled to Cy3 1:600 (Sigma-Aldrich, Louis, MO, C2306).

Seeds were surface sterilized by chlorine gas and sown on 1/2 MS 0.8% agar (w/v) medium supplemented with 1% (w/v) sucrose. After stratification for 2 d in the dark at 4°C, the seedlings were grown at 21°C in a 16-h/8-h d/night cycle for 3–4 d. Seven-day-old seedlings were used for the observation of endocytic foci in roots. Five-day-old seedlings were used for PIN2 Western blot analysis. For the CLC1 PM localization experiment, 4- to 5-d-old seedlings grown under continuous light were used.

All the treatments were carried out at room temperature (RT) by diluting the drugs in liquid 1/2 MS medium containing 1% (w/v) sucrose to the working concentrations. Throughout the imaging time course, the seedlings were kept in treatment conditions, except for the FM4-64 internalization and CLC1 PM localization experiments. For the latter, imaging was done with seedlings settled flatly on a solid agar block containing the drugs dissolved to the working concentration.

BFA treatments: For Supplemental Figures S1, B–D and S2, B, seedlings were pre-treated with either DMSO, 10 μM NAA (10 mM stock), or 10 μM IAA (10 mM stock) for 30 min and then co-treated with 37.5 μM BFA (50 mM stock) and mock or the original auxin for 60 min. In Figure 1, seedlings were pre-treated with ethanol, DMSO, 50 nM (100 µM stock), 10 μM (10 mM stock), or 20 μM (10 mM stock) IAA or NAA for 30 min and then co-treated with 50 μM BFA for 30 min. 10 µM CHX (10 mM stock) treatment was present throughout the experiments. In Supplemental Figure S2E, seedlings were pre-treated with either DMSO, 20 μM NAA (10 mM stock), or 210 μM IAA (10 mM stock) for 30 min and then co-treated with 50 μM BFA (50 mM stock) and mock or the original auxin for 60 min. FM4-64 staining: seedlings were stained with 2 μM FM4-64 in liquid 1/2 MS medium. The seedlings were incubated for 2 min in the dye and washed twice before imaging. For Figure 2, A and B; Supplemental Figure S3A, seedlings were pre-treated with DMSO, ethanol, 10 μM or 100 μM IAA (10 mM stock), or varying concentrations of NAA for 30 min. Blebbistatin treatment: seedlings were pre-treated with DMSO or 500 μM Blebbistatin (1 M stock) before imaging. In Figure 4I and J, roots were pulse-treated with 200 nM (200 µM stock) pep1 for 1 min and then treated with 20 µM NAA (10 mM stock) or the corresponding mock for 60 min. As control, seedlings were not pep1 pulsed (untreated). In Supplemental Figure S5F, roots were pre-treated with 10 μM NAA, 10 μM IAA (10 mM stock), or DMSO (control) for 30 min before the pep1 pulse, followed by the same post-treatment for 60 min. As control, seedlings were incubated with DMSO/mock throughout pep1 pulse (untreated). For PIN2 Western blots, seedlings were treated with 1 µM IAA (1 mM stock) or the corresponding mock for varying durations. For β-estradiol induction, 2-d-old seedlings were transferred to plates containing 2 μM β-estradiol for 24 h. The seedlings were maintained continuously under chemical induction during subsequent imaging. Mock treatments in all experiments contained an equivalent amount of solvent in the treatment conditions.

For immune-staining of the roots, the InsituPro VSi robot was used as described previously (Sauer et al., 2006). Used antibodies are described in the section ‘Reagents used’.

Seedlings on plates were treated by spraying them with liquid 1/2 MS medium containing DMSO (mock) or 1 μM IAA. At the indicated time intervals, roots were harvested and flash frozen in liquid nitrogen. These root samples were ground using a Retsch mill for 2× 1 min at 20 Hz and the resulting root powder was re-suspended in a 1:1 (w/v) ratio of protein extraction buffer (50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% (v/v) Triton X-100, 1× Roche complete™ Mini Protease Inhibitor Cocktail, 1× Roche PhosSTOP^TM, 1 mM EDTA, 1 mM DTT, 10 µM MG-132, and 0.5 mM PMSF (Phenylmethylsulfonylfluorid)). The samples were incubated on ice for 30 min, with intermediate vortexing to mix root powder and extraction buffer, followed by a centrifugation step at 10,000g to sediment the plant debris. The cleared supernatant containing the proteins of interest was collected and the total protein content was determined using Quick Start Bradford reagent (Bio-Rad, Hercules, CA). The protein extracts were all diluted in extraction buffer to the same concentration (30 µg/25 µL) to allow equal loading of the samples. Proteins were separated by SDS–PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) in a 12% (v/v) acrylamide gel (Protean^® TGX^TM, Bio-Rad, Hercules, CA) and were transferred to PVDF (Polyvinylidenfluorid) membranes by electroblotting (wet-transfer, Towbin transfer buffer, Bio-Rad System, Hercules, CA). The membranes were then incubated in blocking buffer (0.05% (v/v) Tween-20, 5% (w/v) milk powder or 3% (w/v) BSA, 20 mM Tris–HCl (pH 7.5), 150 mM NaCl) for at least 60 min and reacted with anti-PIN2 or anti-actin antibodies in TBS-T buffer + 3% BSA. This was followed by an anti-rabbit IgG secondary antibody conjugated to HRP incubation and chemiluminescence reaction. To allow multiple antibody detections using the same PVDF membrane, mild stripping was performed using 15 g L⁻¹ glycine, 1 g L⁻¹ SDS, 10 mL L⁻¹ Tween-20 buffer at pH 2.2 for 2–5 min.

GST and GST-PIN1CL recombinant proteins were expressed in bacteria and purified with glutathione sepharose beads, as described previously (Sancho-Andrés et al., 2016). The receptor binding domain (RBD) of Arabidopsis μ2-adaptin was expressed in bacteria as an histidine-tagged protein (His)6×-RBD-µ2-adaptin and purified with a Nickel column. Buffer exchange was performed using a PD-10 column (Amersham Pharmacia Biotech, Little Chalfont, UK) to binding buffer (100 mM Tris–HCl (pH 7.5), 5 mM EDTA, 0.1% (v/v) Triton X-100) as described previously (Sancho-Andrés et al., 2016). Purified µ2-adaptin protein was pre-incubated for 1 h at RT in the absence or presence of 10 μM NAA, 2,4-D, 2-NAA, or BA and then for 2 h at RT with 30 μL glutathione sepharose beads containing GST or GST-PIN1CL, which also had been pre-incubated with or without the respective auxin analogues. The beads were washed three times with 0.5 mL binding buffer and re-suspended in two-fold sample buffer (Laemmli, 1970). The samples were boiled at 95°C for 3 min and subjected to SDS–PAGE and Western blotting with a His-antibody. Each pull-down assay was independently performed three times and similar results were obtained.