Ab124811

Conventional protocols were used for IB with the details available elsewhere. The following primary antibodies were used for IB: anti-HERPUD1 (1:1000, ab150424, Abcam, Cambridge, UK), anti-GCLC (1:1000, ab190685, Abcam), anti-CBS (1:1000, ab140600, Abcam), anti-CTH (1:1000, ab189916, Abcam), anti-SHMT2 (1:1000, #33443, Cell Signaling Technology (CST), Boston, MA, USA), anti-GSS (1:1000, #ab124811, Abcam) or GSS (H-7) (1:500, sc-166882, Santa Cruz, Heidelberg, Germany), anti-MDM2 (1:1000, ab259265, Abcam), anti-SYVN1 (1:1000, #14773, CST), anti-GAPDH (1:1000, #5174, CST). The membranes were incubated with anti-rabbit IgG, HRP-linked antibody (1:2000, #7071, CST) or anti-mouse IgG, HRP-linked antibody (1:2000, #7076, CST) at room temperature for one hour, and visualized using Clarity Western ECL substrate. The relative expression levels of proteins were determined by densitometry using ImageJ software and were normalized to housekeeping protein.

IHC and IF were performed using conventional protocols that are available elsewhere. For IHC, the primary antibody used was anti-RRM2 (Abcam, #ab172476). The tissue microarray was purchased from U.S. Biomax (Rockville, MD, USA). The final results were confirmed by two independent pathologists. The specimens were scored as follows: the one in which 0% of cells showing signals for staining was tagged as negative (−), 0–10% of cells was tagged as weak positive (−/+) and 11–100% of cells was tagged as strong positive (+). For IF, the primary antibodies used were anti-GSS (Abcam, #ab124811), anti-PSMB5 (Abcam, # ab167341) and anti-RRM2 (Abcam, #ab172476).