Western blotting

Sample buffer (50 mM Tris-HCl pH 6.8, 10% (w/v) glycerol, 4% (w/v) SDS, 0.004% (w/v) bromophenol blue) with 10% (v/v) 2-mercaptoethanol (Sigma-Aldrich) was added to samples, which were then incubated at 95 °C for 10 minutes. The samples were run alongside Precision Plus Protein All Blue Standards (Bio-Rad Laboratories) on 4–12% SDS polyacrylamide gels (Life Technologies) at 100 V. The gels were transferred to nitrocellulose membrane at 30 V for 75 minutes. Membranes were incubated with a casein-based blocking buffer (Sigma-Aldrich) or 5% BSA in Tris-buffered saline with 0.1% Tween-20 (TBST; for phospho-specific antibodies) for 45 minutes. The membranes were incubated with primary antibodies or Alexa Fluor 680–conjugated streptavidin (1:2000; Invitrogen) in blocking buffer overnight at 4 °C.

Proteins levels were analysed by western blotting using the following primary antibodies: pSTAT5 (Y694, #9351, CST); pERK1/2 (#4370, CST), ERK1/2 (#9102, CST), ERK2 (sc-154) STAT5a (sc-1081) and Rab5b (sc-598, Santa Cruz Biotechnology); Prl (ab960, Millipore); Caveolin 1 (#610060, BD Biosciences); Clathrin heavy chain (ab172958), α-tubulin (ab6160, Abcam) and AP2M1 (mouse anti-AP50 Clone 31, BD Transduction Labs). Secondary antibodies used were: anti-rabbit Alexa Fluor 680 (A-21109), anti-mouse Alexa Fluor 680 (A-21058), and anti-rat Alexa Fluor 680 (A-21096, Invitrogen); anti-mouse DyLight 800 (#5257, Cell Signaling); anti-rat Alexa Fluor 800 (STAR71D800GA, AbD Serotec). Fluorescent secondary antibodies were detected by Odyssey scanner (Li-Cor), and intensity of bands quantified using ImageJ.