Ab121190

TNBC sections were deparaffinized and rehydrated with graded ethanol, soaked in EDTA (1 mmol/L, pH 8.0), and then heated to 121 °C in an autoclave for three min to retrieve the antigen. After natural cooling and rinsing with phosphate-buffered saline (PBS, pH 7.2), 0.3% hydrogen peroxide was applied for 20 min to block endogenous peroxide activity. Thereafter, 10% goat serum was applied for 1 h at room temperature to block any nonspecific reactions. After washing with PBS (pH 7.2), the sections were incubated with a rabbit anti-RPLP1 polyclonal antibody (diluted 1:100; Abcam, ab121190, USA) for 2 h. Negative control slides were processed in parallel using a nonspecific IgG antibody (diluted 1:100; Abcam, USA) at the same concentration as the primary antibody. All sections were processed using the peroxidase-anti-peroxidase kit according to the manufacturer’s instructions (Dako, Germany).

Cells were promptly homogenized in lysis buffer and then centrifuged at 13,000g for 20 min at 4 °C. The supernatant was diluted twofold in SDS loading buffer and denatured at 100 °C for 15 min. An equivalent amount of protein from each sample was loaded onto a 10% SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, USA). The membranes were incubated overnight at 4 °C with the primary antibodies. The antibodies were as follows: anti-RPLP1 (1:500, ab121190, Abcam, Cambridge, MA, USA), anti-E-cadherin antibody (1:1000, ab1416, Abcam), anti-vimentin antibody (1:1000, ab92547, Abcam), anti-N-cadherin antibody (1:1000, ab18203, Abcam), anti-Snail antibody (1:1000, ab180714), and anti-β-actin (1:5000; Abcam). After washing three times with tris-buffered saline with 0.1% tween-20 (TBST) for 5 min each time, the membranes were then incubated with horseradish peroxidase-conjugated secondary human anti-mouse or anti-rabbit antibodies (1:2000; Abcam) for 2 h at room temperature. The bands were then detected using an enhanced chemiluminescence detection system (Bio-Rad, USA).