Sab4300645 100ug

Following transfection, the cells were washed three times with PBS and lysed with radioimmunoprecipitation assay lysis buffer to obtain the total protein. Protein concentration was measured using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.). Total proteins (50 µg) were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride membranes, as previously described (20 (link)). The membranes were incubated with blocking buffer (5% skimmed milk) for 1.5 h at room temperature and then incubated with the following primary antibodies: anti-CDK6 (1:500, ab79454; Abcam, Cambridge, MA, USA) and anti-GAPDH antibody (1:500; SAB4300645-100UG; Sigma-Aldrich, Merck KGaA) at 4°C overnight. Following incubation with the primary antibody, blots were washed with TBS-0.1% Tween and subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit (1:2,000, ab6721) or rabbit anti-mouse IgG (1:2,000, ab6709) (both from Abcam) secondary antibodies at 37°C for 2 h. Band intensity was quantified using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore, Billerica, MA, USA). Experiments were performed in triplicate.

Proteins were extracted from the cell lines using Radio-immunoprecipitation Assay lysis buffer (Thermo Fisher Scientific, Inc.) for 20 min at 4°C with occasional agitation. Equal amounts of protein extracts (determined using the BCA method) were subjected to 10% SDS-PAGE and subsequently transferred to a polyvinylidene fluoride membrane (0.45 µm pore size; EMD Millipore, Billerica, MA, USA). Followed by blocking with 5% skimmed milk for 1.5 h at room temperature, the membrane was incubated with primary antibodiesanti-PRL-3 (dilution, 1:500; cat. no. ab50276; Abcam), anti-p53 (dilution, 1:500; cat. no. ab31333; Abcam), anti-p14^ARF (dilution, 1:500; cat. no. ab3642; Abcam) and anti-GAPDH antibody (dilution, 1:500; cat. no. SAB4300645-100UG; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 4°C overnight and rinsed twice with PBS, followed by incubation with peroxidase-conjugated secondary anti-rabbit (dilution, 1:2,000; cat. no. ab6721; Abcam) or anti-mouse IgG antibodies (dilution, 1:2,000; cat. no. ab6709; Abcam) for 1 h at room temperature. The membrane was subsequently incubated with an enhanced chemiluminescent substrate kit (Thermo Fisher Scientific, Inc.) and exposed to X-ray film. ImageJ (version 1.41; National Institutes of Health, Bethesda, MD, USA) was used for the quantitative analysis of band densities. GAPDH was used as a control (primers as aforementioned).