Sc 374573

Primary antibodies against OGT (24083) and O-GlcNAc (9875) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against β-actin (A300–491A) were purchased from Bethyl Laboratories (Montgomery, TX, USA), and those against PRKCG (SC-166451) and PDGFRB (SC-374573) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Secondary antibodies against horseradish peroxidase-linked anti-rabbit (A120–101P) and anti-mouse (A90–116P) antibodies were purchased from Bethyl Laboratories. For immunohistochemistry (IHC), primary antibodies against OGT (ab96718) and Ki67 (ab15580) were purchased from Abcam (Cambridge, UK), and antibodies against Caspase-3 (9661) were purchased from Cell Signaling Technology. GEM (G6423) was purchased from Selleck Chemicals (Houston, TX, USA). PTX (T7402), Thiamet G (SML0244), MG132 (M7449), and phosphatase inhibitor cocktails 2 and 3 were purchased from Sigma-Aldrich. MTT (M1415) was purchased from Duchefa Biochemie (Haarlem, The Netherlands). Protease-inhibitor cocktail tablets were purchased from Roche Applied Biosciences, and RNAi-Max (13778150) was purchased from Thermo Fisher Scientific.

Mouse kidney sections or treated NRK-49F cells on slides were fixed in 4% paraformaldehyde solution at 4 °C overnight, and then 0.2% Triton X-100 was applied for permeabilization of the cell membrane. After 15 min permeabilization, the slides were blocked by 5% bull serum albumin (BSA) at 37 °C for 2 h. The slides were incubated with primary antibody at 37 °C for 2 h, and then washed by PBS at room temperature for 3 times × 5 min. Then the slides were incubated with the fluorescein isothiocyanate (FITC)-conjugated secondary antibody (A21203, A21206; Invitrogen, Waltham, MA, USA) at 37 °C for 1 h. After washing with PBS, the slides were stained by 4′6-diamidino-2-phenylindole (DAPI, D9542-10MG; Sigma-Aldrich, St Louis, MO, USA) for 2 min, and observed with a microscope equipped with a digital camera (IX51 + DP72; OLYMPUS, Tokyo, Japan) after washing. The antibodies against S100A16 (11456-1-AP; Proteintech, Chicago, IL, USA), PDGFRβ (sc-374573; Santa Cruz Biotechnology, Dallas, TX, USA), β-catenin (610153; BD Biosciences, San Jose, CA, USA), HRD1 (13473-1-AP; 67488-1-Ig; Proteintech, Chicago, IL, USA), GSK3β (9832S; Cell Signaling Technology, Danvers, MA, USA), CK1α (ab206652; Abcam, Cambridge, UK), were used with 1:100 dilution. The FITC-conjugated secondary antibodies were diluted in 1:1000, and the DAPI was diluted in 1:10,000.