Goat anti rabbit secondary antibody

Radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (BestBio Cat No. BB-3101) was used to extract proteins. The protein concentration was assessed using the Rapid Gold BCA Protein Assay Kit (Thermo Fisher). Western blotting analysis was performed by loading 15 µg of lysate onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferring the gels to polyvinylidene difluoride (PVDF) membranes (Millipore), and incubated with rabbit anti-GDF5 (1:1000, #A13167; ABclonal), rabbit anti-PPARγ (1:1000, #2443; CST), rabbit anti-FASN (1:1000, #D262701; Sangon Biotech), rabbit anti-C/EBPα (1:1000, #2295; CST), rabbit anti-FABP4 (1:1000, #2120; CST), rabbit anti-CD36 (1:1000, #ab1336-25; Abcam), rabbit anti-GAPDH (1:5000, #BS65529; Bioworld), rabbit anti-CD9 (1:1000, #AP68-965-100; Abcepta), rabbit anti-CD63 (1:2000, #D160973; Sangon Biotech), rabbit anti-TSG101 (1:2000, #381538; ZEN BIO), rabbit anti-Alix (1:1000, #D262028; Sangon Biotech) or rabbit anti-Calnexin (1:1000, #D262986; Sangon Biotech). Afterward, goat anti-rabbit secondary antibody (1:50000, # BS13278, Bioworld) conjugated with HRP was used. GAPDH levels served as the loading control. The amount of protein was measured using ImageJ software.

Tissues and cells were homogenized in whole-cell lysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl and 1.0% NP-40) containing a protease inhibitor cocktail and 20 mM N-ethylmaleimide to prevent SUMO de-conjugation. Immunoblotting was performed with primary antibodies against HOXA10 (1 : 1000; Santa Cruz, Santa Cruz, CA, USA), SUMO1 (1 : 1000; Abcam, Cambridge, CA, USA), SENP1 (1 : 1000; Abcam), SENP2 (1 : 1000; Abgent, San Diego, CA, USA), Flag (1 : 1000; Sigma), Myc (1 : 5000; Invitrogen, Carlsbad, CA, USA), Lamin B1 (1 : 1000; Bioworld, St Louis Park, MN, USA), HSP90B (1 : 1000; Bioworld), ubiquitin (1 : 1000, Abcam), acetylated lysine (1 : 1000; CST, Danvers, MA, USA) and GAPDH (1 : 10 000; Bioworld), followed by donkey anti-goat or goat anti-rabbit secondary antibody conjugated with HRP. Detection was performed using an enhanced chemiluminescence kit (Millipore, Billerica, USA).