Hrp conjugated goat anti mouse antibody

Cell lysates were prepared with RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors. Equal amounts of cell lysates were fractionated with SDS-PAGE and then blotted onto nitrocellulose membranes. The membranes were blocked for 1 h at room temperature with 5% skim milk and then probed with mAb 1D7 supernatant for 2 h at room temperature. After the membranes were washed with TBST (10 min each), they were incubated with an HRP-conjugated goat anti-mouse antibody (1:6000; Proteintech) for 1 h at room temperature. After the membranes were washed three times, the signals were visualized with an ECL kit (Thermo Fisher) and detected with the Tanon 5200 Chemiluminescent Imaging System.

Western blot assay were conducted according to previous description^{32 (link)}. The primary antibodies used include the following: anti-MCPIPI (#GTX110807, GeneTex, 1:1500), anti-cyclin D1 (#A19038, ABclonal, 1:1000); anti-RB (#25628–1-AP, Proteintech, 1:1000), anti-pRB(Ser780) (#9307, Cell Signaling Technology, 1:1000), anti-E2F1 (#12171–1-AP, Proteintech, 1:1000), anti-Flag-Tag (#T0003, Affinity Biosciences, 1:5000), anti-GAPDH (#60004–1-Ig, Proteintech, 1:5000), anti-β-actin (#60008-1-Ig Proteintech, 1:1000). The secondary antibodies used include HRP-conjugated goat anti-mouse antibody (#SA00001-1, Proteintech, 1:10,000) and HRP-conjugated goat anti-rabbit antibody (#SA00001-2, Proteintech, 1:10,000). The bands were detected with ECL reagent (Advansta, USA). Image J software was used to calculate the protein expression levels.