Anti ccnd1

Cells were lysed with RIPA buffer (Beyotime, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) for 10 min on ice and centrifuged at 14,000 × g. The supernatants were collected, and whole cell extracts were separated by 10% SDS-PAGE gel and transferred onto a PVDF membrane (GE, USA). The membranes were blocked with 5% skimmed milk (BD, USA) in TBST buffer and incubated with primary antibodies at 4 °C overnight. Primary antibodies used were anti-IRS2 (1:1,000, Proteintech), anti-CCND1 (1:1,000, Servicebio, China), anti-ERK1/2 (1:1,000, CST, USA), anti-p-ERK1/2 (1:1,000, Proteintech, China), and anti-β-actin (1:1,000, Sigma, USA). After washing with PBS five times, the membranes were incubated with near-infrared fluorescent anti-mouse (1:50,000, LI-COR, USA) or anti-rabbit (1:25,000, Jackson, USA) secondary antibodies for 1 h at room temperature. Fluorescent signal was detected by Odyssey Near-infrared fluorescence imaging system (LI-COR, USA).

Immunofluorescence assay was performed as previous described 42 . Briefly, tumor tissue was fixed in 4% paraformaldehyde for 24 h following by dehydration with 30% sucrose and frozen in O.C.T. Compound (Fisher Scientific, USA), cryosectioned at 8 μm, and mounted on slides. Tissue sections were permeabilized with PBS containing 0.3% Triton X-100 and blocked in PBS containing 1% BSA at 4 °C for 1 h, and then incubated with primary antibodies in PBS at 4 °C overnight, followed by three washings in PBS and further incubation with Cy3-conjugated secondary antibodies (1:300, Proteintech, China) at room temperature for 1 h and by another three washings. The primary antibodies used were anti-IRS2 (1:100, Proteintech, China), anti-CCND1 (1:100, Servicebio, China), anti-Ki67 (1:500, Proteintech, China). Sections were stained with DAPI and mounted using antifade mounting medium (VECTASHIELD, USA), photographed with a fluorescence microscope.