Otl2b8

IHC was performed using an immunohistochemistry kit according to the manufacturer’s instructions (E-IR-R211; Elabscience, Wuhan, China). The specimens were scored according to the percentage of positively stained cells (0 = negative; 1 = 1–10%; 2 = 11–50%; 3 >50%) and intensity of staining (0 = no staining; 1 = slight staining; 2 = moderate staining; 3 = strong staining). The final scores were obtained by multiplying the percentage of positive cells by the staining intensity. Specimens with scores 0–3 and 4–9 were assigned to the low-and high-expression groups, respectively. Anti-HAPSTR1 (1:100, OTl2B8; Origene, Rockville, MD, USA), anti-LRPPRC (1:100, 21175-1-AP; Proteintech, Rosemont, IL, USA), anti-Beclin1 (1:100, WL02508; Wanleibio, Shenyang, China), and anti-Ki-67 (1:500, WL0280a; Wanleibio) antibodies were used.

Total protein was extracted using a cell lysis buffer for Western blotting and immunoprecipitation (Beyotime, Shanghai, China) containing PMSF and a protease inhibitor cocktail. Proteins (30 μg/well) were separated using 10% SDS/PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The primary antibodies used in this study were against anti-HAPSTR1 (1:1000, OTl2B8; Origene), anti-HAPSTR1 (1:500, OTI2D1; Novusbio, Centennial, CO, USA), anti-LRPPRC (1:5000, 21175-1-AP; Proteintech), anti-β-actin (1:1000, 20536-1-AP; Proteintech), anti-Ub (1:1000, 10201-2-AP; Proteintech), anti-N-cadherin (1:5000, 66219-1-Ig; Proteintech), anti-Beclin1 (1:2000, T55092; Abmart, Berkeley Heights, NJ, USA), anti-P62/SQSTM1 (1:2000, T55546; Abmart), anti-ATG5 (1:2000, T55766; Abmart), anti-vimentin (1:500, WL01960; Wanleibio), anti-snail (1:500, WL01863; Wanleibio), and anti-E-Cadherin (1:500, WL01482; Wanleibio).