Mouse anti flag

All the fragments were ligated into pcDNA3.1(+) plasmid, and the constructed plasmids were then verified by DNA sequencing. The pCre-luc (Santa Cruz, CA, USA) was kindly gifted by Xin Xie’s Lab of Tongji University. Human α-melanocyte stimulating hormone (α-MSH) and human adrenocorticotropin ACTH (1–24) was synthesized by Genescript (Nanjing, China). We purchased the paraformaldehyde (4%), phosphate buffered saline (PBS), bovine serum albumin, non-fat milk powder and β-mercaptoethanol from Sangon Biotech (Shanghai, China). TRNzol Universal Reagent and FastQuant RT Kit (with gDNase) were obtained from Tiangen Biotech (Beijing, China). Tetramethylbenzidine (TMB) chromogen solution was purchased from Beyotime^® Biotechnology (Shanghai, China). Hydrochloric acid and sulfuric acid were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). The antibodies used in this study included Rabbit anti-Flag (Cell Signaling Technology, USA), Mouse anti-HA (Sigma Aldrich, MO, USA), Mouse anti-Flag (Abcam, Cambridge, UK), Goat anti-Mouse IgG (horseradish peroxidase (HRP)-conjugated) (ABclonal Biotech Co., Ltd, Wuhan, China), and Goat Anti-Rabbit Alexa-Fluor 594 (Abcam). All primers used for full-length gene amplification, reverse transcription PCR (RT-PCR), and real-time quantitative PCR are listed in Supplemental Table 1 and synthesized by GENEWIZ (Suzhou, China).

Triton X‐100 lysis buffer [20 mm Tris (pH 7.4), 2 mm EDTA, 150 mm sodium chloride, 1 mm sodium deoxycholate, 1% Triton X‐100, 10% glycerol, 2 pills protease inhibitor cocktail (Roche)] was used to collect protein from cells. Protein samples were heat‐denatured and equally loaded, separated on 8–12% SDS/PAGE gel, transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK), and blocked with 5% nonfat dry milk. Primary antibodies for western blot analyses included mouse anti‐α‐tubulin (1 :  20 000 dilution; Sigma), rabbit anti‐Snail (1 : 1000 dilution; Cell Signaling, Danvers, MA, USA), rabbit anti‐Slug (1 : 1000 dilution; Cell Signaling), rabbit anti‐CHIP (1 : 1000 dilution; Abgent, San Diego, CA, USA), mouse anti‐Flag (1 : 3000 dilution; Abcam), mouse anti‐GFP (1 : 1000 dilution; Santa Cruz), mouse anti‐HA (1 : 1000 dilution; Abcam, Cambridge, MA, USA), mouse anti‐Vimentin (1 : 1000 dilution; Santa Cruz, Santa Cruz, CA, USA), mouse anti‐E‐cadherin (1 : 5000 dilution; BD Biosciences, San Jose, CA, USA), and mouse anti‐Ub (1 : 5000 dilution; Santa Cruz). Membranes were incubated with horseradish peroxidase‐conjugated anti‐mouse or anti‐rabbit secondary antibody (1 : 5000 dilution; Cell Signaling) for 1 h, and chemiluminescence signals were detected by ECL substrate (GE Healthcare, Chicago, IL, USA).