Immunoblotting and immunoprecipitation

For western blotting, cells were washed twice in PBS, harvested in 1% NP-40 Lysis Buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1X Protease Inhibitor Cocktail and 0.5 mM DTT) and incubated for 30 min at 4°C. Extracts were cleared by centrifugation at 14,000 x g at 4°C for 45 min and analyzed. The supernatant was removed, and protein concentrations were determined by bicinchoninic acid (BCA) assay (Pierce). Equal amounts of total protein from each sample were analyzed by SDS-PAGE on Criterion gels (Bio-Rad). SDS-PAGE samples were transferred onto Protran Nitrocellulose (GE) or Immobilon-P PVDF (Millipore) using Tris/Glycine/SDS Buffer (Bio-Rad) at 100V for one hour at 22°C. Membranes were blocked with 5% milk and 0.1% Tween-20 in Tris-buffered saline (TBS) for 1 hr at room temperature. Membranes were immunoblotted for 1 hr at 22°C using the following antibodies: rat anti-HA (1:3000; Roche), mouse anti-V5 (1:5000; Life Technologies), rabbit anti-SHH (H-160) (1:1000; Santa Cruz), rabbit anti-GFP (1:8000, Rockland), rabbit anti-Kinesin (anti-Kif5B, 1:5000; Abcam), mouse anti-α-Tubulin (DM1A) (1:5000, CST), followed by three 5 min washes in secondary milk (primary milk diluted to 25% with TBS). Corresponding HRP-conjugated secondary antibodies (Jackson Immuno) were incubated for 1 hr at RT at a 1:5000 concentration. Blots were developed using an Odyssey Fc (Li-Cor) with ECL Prime (GE).

For immunoprecipitation assays, proteins of interest were expressed in NIH3T3 cells. Cell lysates were prepared ~48 hr post-transfection using a 0.5% NP-40 Lysis Buffer (30 mM Tris-HCl, pH 7.4, 75 mM NaCl, 0.5% NP-40, 5% glycerol, 2 mM MgCl2, 4 mM KCl, 1 mM EDTA, and 1X Protease Inhibitor Cocktail) and incubated for 30 min at 4°C with two units per mL of Benzonase Nuclease to degrade DNA from protein samples. Extracts were cleared by centrifugation at 14,000 x g at 4°C for 30 min, supernatant was collected, and protein concentration was determined by BCA assay (Pierce). Equal total protein amounts for each sample were used in co-immunoprecipitation assays and analyzed by SDS-PAGE on Criterion gels (Bio-Rad). Co-immunoprecipitation assays were performed as described (Stewart et al., 2018) with the following modifications. Samples were pre-cleared with A/G Plus Agarose for 30 min with gentle rotation. Samples were centrifuged at 1000 x g for 1 min and set up in new tubes with either anti-Mouse IgG1 control or EZview Red Anti-Flag Affinity Gel (Sigma) (to immunoprecipitate Flag epitope-tagged proteins) for three hours at 4°C with gentle rotation. Samples were then centrifuged at 1000 x g for 1 min and supernatant was removed. Beads were washed 3x for 5 min each with 0.5% NP-40 Lysis Buffer with gentle rotation at room temperature. Proteins were eluted from agarose beads with 1X SDS sample buffer (2% SDS, 4% v/v Glycerol, 40 mM Tris-HCl, pH 6.8, 0.1% Bromophenol blue) by incubating them at room temperature for 5 min. Samples were centrifuged at 2000 x g for 2 min and the eluent was transferred to a new tube. Immunoprecipitates were analyzed by western blot using the following antibodies: rabbit anti-GFP (1:8000; Rockland), rabbit anti-Flag (DDDDK) (1:3000, Abcam), rabbit anti-Shh (1:2000; SCBT), and rabbit anti-Kif5B, (1:5000; Abcam).