Pan acetylated lysine

The western blot primary antibodies against pan-Acetylated lysine (9441, 1:1000), SIRT3 (2627, 1:1000), Myc (2272, 1:1000) were purchased from Cell Signaling Technology. Antibody against Flag (SAB4301135, 1:5000) was purchased from Sigma-Aldrich. Antibody against Tubulin (AT819-1, 1:5000) were purchased from Beyotime. Antibody against MTHFD2 (sc-390708, 1:5000) was purchased from Santa Cruz.
To generate a site-specific antibody to detect the acetylated K88 of MTHFD2 (α-acK88, 1:500), synthesized peptide PASHSYVLNK (Ac) TRA (Shanghai HuiOu Biotechnology Co.Ltd) was coupled to KLH as antigen to immunize rabbit. Anti-serum was collected after four doses of immunization.

TMA construction was performed as previously reported [27 (link)]. Staining of LC3B (polyclonal; dilution: 1:500; incubation time: 30 min; NovusBio, Centennial, CO, USA), p62 (Clone: EP396; dilution: 1:200; incubation time: 30 min; Abcam, Cambridge, UK), Beclin-1 (monoclonal; dilution: 1:100; incubation time: 30 min; REF#AC-0276, Abcam, Cambridge, UK) and pan-acetylated-lysine (polyclonal; dilution: 1:1000; incubation time: 30 min; REF#9441, Cell Signaling Technology, Danvers, MA, USA) was conducted. Stained slides were scanned with the Pannoramic slide scanner (3DHISTECH, Budapest, Hungary). Evaluation of the IHC staining was performed semi-quantitatively by determining the percentage of positively stained tumor cells (0: <5%, 1: 5–25%, 2: 26–75%, 3: >75%) and the staining intensity (0: none/background, 1: weak, 2: moderate, 3: strong). Due to stronger background staining for LC3B, IHC scores ≤4 and ≥5 were considered low and high, respectively. For p62, Beclin-1 and pan-acetylated-lysine, IHC scores ≤3 and ≥4 were considered low and high, respectively. IHC analysis was performed by two independent investigators. In line with literature, expression of LC3B high/p62 low was defined as ‘active autophagy’ [22 (link),29 (link)]. TMA cores lacking representative tumor tissue or exhibiting staining artifacts were excluded from the analysis.