Radioimmunoprecipitation assay (ripa)

Total protein was extracted from each of the cell lines (non-transfected, NT or RPLP1 siRNA reverse transfected plated at a density of 3.0 × 10⁵ cells/well) using RIPA buffer (1X RIPA, Catalog #9806, Cell Signaling Technologies [CST], Danvers, MA, USA). The protein concentration in each sample was determined using the Bio-Rad Protein Assay ([Catalog 3500-0006], Bio-Rad Laboratories, Richmond, CA, USA). The same amount of protein (30 μg) was subjected to 12% bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (w/v) gel electrophoresis and electroblotted onto PVDF membranes (Invitrogen). RPLP1 (21636-1-AP; 1:400; Proteintech, Rosemont, IL, USA) and donkey, anti-rabbit secondary antibody (catalog #NA934V; 1:20,000; GE Healthcare/Fisher Scientific, Pittsburgh, PA, USA) were used. Stripping and re-probing for β-actin (ab8227; 1:10,000; Abcam, Cambridge, MA, USA) was conducted to normalize RPLP1 protein expression levels.

Immunoblot was performed as described previously (21 (link)) with minor modifications. Briefly, CD4⁺ T cells were isolated using MACS microbeads (Miltenyi Biotec). For protein isolation, cells were lysed with 1xRIPA (Cell Signaling Technology) supplemented with cOmplete™ Protease Inhibitor Cocktail (Sigma) and 0.1% SDS for 30 min on ice. Proteins were detected using rabbit-anti-mouse Atg7 (Abcam) and rabbit-anti-mouse β-actin (Novus Biologicals) antibodies and visualized using chemiluminescence.

Total protein was extracted from 12Z cells using RIPA buffer (1X RIPA, Catalog #9806, Cell Signaling Technologies (CST), Danvers, MA, USA). Protein concentration in each sample was determined using the Bio-Rad Protein Assay ((Catalog 3500-0006), Bio-Rad Laboratories, Richmond, CA, USA). The same amount of protein (30 μg) was subjected to 12% Bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (w/v) gel electrophoresis and electroblotted onto PVDF membranes (Invitrogen). PTEN (9188; 1:1000; CST) and donkey, anti-rabbit secondary antibody (catalog #NA934V; 1:20000; GE Healthcare/Fisher Scientific, Pittsburgh, PA, USA) were used. Stripping and re-probing for β-actin (ab8227; 1:10,000; Abcam, Cambridge, MA, USA) were conducted to normalize PTEN protein expression levels.

Total cell lysate was obtained by incubating treated and untreated DLD-1 cells in 1X RIPA (Cell Signaling Tech, Beverly, MA, USA), with phosphatase inhibitor cocktail and protease inhibitor tablets (Roche), for 30 min at 4 °C and followed by removal of cell debris by centrifugation at 20,000× g at 4 °C. Protein concentrations were determined by BCA Assay (Pierce, Waltham, MA, USA).

HaCaT cells were lysed using RIPA (radioimmunoprecipitation assay) buffer (Cell Signaling), and centrifuged at 17,000× g for 15 min at 4 °C. Supernatants were collected and protein concentrations were determined using DC protein assay reagents (Bio-Rad Laboratories, CA, USA). Proteins (50–130 μg/lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, St. Louis, MO, USA) at 4 °C. Membranes were blocked in 3% skim milk at RT for 2 h, washed with PBS containing 0.05% Tween-20, incubated with each target primary antibody (1: 1000–10,000 dilution), and then with horseradish peroxidase-conjugated secondary antibody at RT for 1 h. Protein bands were visualized using a chemiluminescence substrate and detected using a chemiluminescence imaging system (LuminoGraph, ATTO, Tokyo, Japan).

EOC cell lines were lysed in 1x radioimmunoprecipitation assay buffer (RIPA, Cell Signaling Technology, BioConcept, Switzerland) containing proteinase inhibitor cocktails (Sigma-Aldrich/Merck, Switzerland). Lysates were clarified by centrifugation at 18,000 × g for 15 min at 4 °C. Clarified lysates were boiled in 1x sample buffer (50 mM Tris-HCl, 1% SDS, 100 mM DTT, and 10% glycerol) at 95 °C for 5 min and resolved by SDS-PAGE. Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad, Switzerland) and blocked with 5% (w/v) bovine serum albumin in TBS-T (20 mM Tris-Base, 150 mM NaCl, pH 7.8, 0.1% Tween 20) for 1 h at room temperature. The membrane was incubated with one of the listed primary antibodies (Supplementary Table 2) diluted in 5% (w/v) BSA in TBS-T overnight at 4 °C. After washing (3 times, 10 min) in TBS-T, membranes were incubated with corresponding HRP-conjugated secondary antibodies (1:10,000, Cell Signaling, BioConcept, Switzerland) for 3 h at room temperature. Finally, the membrane was incubated with Super Signal West Dura Extended Duration Substrate (Thermo Fisher Scientific, Switzerland) for the detection of HRP. Western blot results were visualized by Gel Doc XR + TM (BioRad, Switzerland) and analyzed by Image Lab^TM software (BioRad, Switzerland). Original membranes can be found in Supplementary Fig. 2.