Anti p27kip1

Whole cell lysates were prepared from harvested cells using radioimmunoprecipitation (RIPA) buffer (Life Technologies, Warrington, UK) containing Halt phosphatase and protease inhibitors (Thermo Scientific, Altrincham, UK). The total concentration of protein was determined using Peirce BCA Protein Assay Kit (Life Technologies, Warrington, UK) following the manufacturer’s instructions. The following antibodies were used: anti LC3 (Cell Signaling Technology, Beverly, MA, USA), anti-p62 (Cell Signalling Technology), anti-p53 (DO1) (Santa Cruz, Dallas, TX, USA), anti-p27^kip1 (Santa Cruz, Dallas, TX, USA), anti-pγH2AX (Cell Signalling Technology) and anti-GAPDH (Origene, Cambridge, UK). Densitometry was performed using UVP imaging software to quantify relative amounts of proteins detected on Western blot membranes. Detailed information can be found in Supplementary Figure S3.

The cells were lysed with RIPA buffer (Sigma-Aldrich) for 30 min at 4 °C and centrifuged at 9000 rpm for 20 min at 4 °C. The protein concentration was measured using the micro-BCA assay kit (Invitrogen). Next, the protein samples were denatured at 95 °C for 5 min and were loaded at 20 µg per well along with 5 µL of prestained protein marker (Nippon Gene, Tokyo, Japan) into 10–20% or 15% mini polyacrylamide gel (Atto, Tokyo, Japan) in a reduced condition. The gel was then transferred onto a PVDF membrane (Invitrogen) using a semi-dry blotting method. The membranes were blocked in 3% skim milk (Sigma-Aldrich) before incubation with the following antibodies: anti–p27/Kip1 (Santa Cruz Biotechnology), anti–p53 (Santacruz Biotechnology), anti–p21 (Santacruz Biotechnology), anti–HIF1α antibody (R&D Systems, Minneapolis, MN, USA), anti–Ki67 antibody (R&D Systems), and an anti–GAPDH (R&D Systems). The membranes were then conjugated with anti–HRP secondary antibodies. Before detecting protein bands using a luminescent gel imager (LumiCube; Liponics, Tokyo, Japan), the membrane was incubated with ECL western blotting detection reagents (Millipore Sigma, Burlington, MA, United States) for 5 min in the dark. Finally, the protein bands were quantified using ImageJ software by normalizing the band intensity of each protein marker to the GAPDH protein band as the control.

Purified pretumor B cells or tumor cells were lysed in radioimmunoprecipitation assay lysis buffer with protease and phosphatase inhibitor cocktails. Lysates were separated by SDS-PAGE (Bio-Rad). Protein was transferred from the gel to a nitrocellulose membrane (Bio-Rad). Membranes were probed with anti-p27^Kip1 (Santa Cruz) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam) primary antibody and then incubated with IRDye secondary antibodies (LI-COR Biosciences). Protein bands were visualized with an Odyssey Imager and analyzed with Image Studio (LI-COR Biosciences).