Minichemi chemiluminescence imager

According to the results of the CCK-8 and colony formation assays, we found that the sensitivity of SLC7A11 overexpressed cells to paclitaxel was significantly enhanced when the concentration of paclitaxel reached 15.625 nmol/L. Therefore, the cells were treated with 0, 15.625, or 31.25 nmol/L paclitaxel for 72 hours to analyze the effect of paclitaxel on the expression levels of related proteins by western blotting. RIPA protein lysis buffer (Beyotime Inst Biotech, Shanghai, China) and a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) were used to extract and quantify the proteins. The antibodies were used as follows: SLC7A11 (ab37185, Abcam, Cambridge, UK), p21 Waf1/Cip1 (CST #2947, Cell Signaling Technology, Boston, Mass, USA), p27 Kip1 (CST #3686), CDK2 (CST #2546), CDK7 (CST #2916), Cyclin A2 (CST #4656), Cyclin B1 (CST #12231), Cyclin D3 (CST #2936), LC3 A/B (CST #12741), STX17 (YN4187, Immunoway, Plano, TX, USA), RAB33B (YN1177, Immunoway), UVRAG (CST #13115), Atg 7 (CST#8558), Atg16L1 (CST #8089), Akt (CST #4691), β-Tubulin (CST #2128), GAPDH (CST #5174), and anti-rabbit IgG, and an HRP-linked secondary antibody (CST #7074). Finally, the blots were visualized using the chemiluminescent substrate (ECL) (34580, Thermo Fisher Scientific) and the MiniChemi™ Chemiluminescence imager (Sage Creation, Beijing, China).

For immunoblotting, samples were lysed in 2× SDS-loading buffer and boiled for 3 min at 95 °C. Proteins were separated by SDS-PAGE on Precast 4–12% Bis-Tris gels (Biofuraw, 180-8008H) and transferred onto nitrocellulose membrane with 0.2 μm pore size (GE Healthcare, 10600001). After three washes in TBST (150 mM NaCl, 50 mM Tris-HCl, 0.05% Tween 20, pH 7.5), the membrane was incubated in blocking buffer (5% skimmed milk powder in TBST) for 1 h, followed by incubation with primary antibodies in antibody dilution buffer (4% BSA in TBST) overnight at 4 °C. After three washes in TBST for 15 min each, the membrane was incubated with HRP-conjugated secondary antibodies in antibody dilution buffer for 1 h and washed for 15 min three times. Western Lightning Plus-ECL (PerkinElmer, NEL104001EA) was used to detect proteins on membranes, and the luminescent signals were captured with X-ray films (Fig. 1c) or MiniChemi Chemiluminescence imager (Sagecreation) (Figs. 4f, 6c, 7b and Supplementary Fig. 1a). After the detection of IFT81 and RPL4 (Fig. 1c), the immunoblots were stripped off previous antibodies by incubating in stripping buffer (EpiZyme, PS107) for 15 min and used to detect eIF3d and eIF3h, respectively.
Antibodies for immunoblotting were listed in Supplementary Table 3.