Anti clathrin

The following antibodies were used as markers of subcellular compartments: anti-Rab4 (BD Biosciences Catalog Number 610888, Antibody registry ID AB_398205), anti-Clathrin (BD Biosciences Catalog Number 610499, Antibody registry ID AB_397865), anti-p47a/AP3M1 (BD Biosciences Catalog #610900, AB_10015260) and anti-adaptin β (BD Biosciences Catalog #610381, Antibody Registry ID AB_397765). We used anti-GFAP antibodies (Advanced Immunochemical Incorporated Catalog #031223—Lot 1gf, Antibody registry ID AB_2314538). Antibodies were used at 1:250–500 dilutions.

CADA hydrochloride was synthesized as described previously [76 (link)]. CADA was dissolved in dimethyl sulfoxide (DMSO) and stored at a stock concentration of 10 mM at room temperature. Western blot and flow cytometry antibodies were purchased from (i) Genscript (Piscataway, NJ, USA): anti-V5 (cat #A01724); (ii) BD Biosciences (Allschwil, Switzerland): anti-clathrin (cat. #610500); (iii) Thermo Fisher Scientific (Waltham, MA, USA): anti-β-actin (cat. #MA1-140) and allophycocyanin (APC)-labelled anti-mouse CD4 (clone GK1.5; cat. #47-0041-82); (iv) Sigma (Saint Louis, MO, USA): anti-FLAG (cat. #F1804); (v) Cell Signaling Technology (Danvers, MA, USA): anti-DNAJC3 (p58IPK) (cat. #2940S); (vi) Dako (Santa Clara, CA, USA): HRP-labelled goat anti-mouse immunoglobulins (cat. #P0447) and HRP-labelled swine anti-rabbit (cat. #P0399).

Standard recombinant DNA techniques were used to construct pCS2/FLAG-rat Fz2 (FLAG-Fz2) and pPGK-neo/Wnt5a. pSuper-retro-GFP-Neo-shWnt5a, which was used for the generation of cells stably expressing Wnt5a shRNA, was generated by inserting small hairpin RNA against Wnt5a (5′-GTGGATAACACCTCTGTT-3′) into the pSuper-retro-GFP-Neo vector (Oligo Engine, Seattle, WA). The small interfering RNAs (siRNAs) used in this study are listed in Table S1. Wnt5a was purified to near homogeneity using three chromatography steps as described previously16 (link)55 (link). Anti-Wnt3a, anti-Wnt5a/b, anti-Src, anti-Fyn, anti-Yes, anti-phospho-Src family (Tyr416), anti-AKT, anti-phospho-AKT (Ser473), anti-phospho-PKC (pan) (Ser660), anti-SAPK/JNK, and anti-phospho-SAPK/JNK (Thr183/Tyr185) antibodies were purchased from Cell Signaling Technology (Beverly, MA). Anti-Rac1, anti-EEA1, anti-HSP90, anti-PKCα, and anti-Clathrin antibodies were from BD Biosciences (San Jose, CA). Anti-FLAG M2 and anti-Ror2 antibodies were from Sigma-Aldrich (Steinheim, Germany) and R&D Systems (Minneapolis, MN), respectively.