Ptm 1406

Protein detection was performed by incubating MCT1 (1:1000; AB90582, Abcam, Cambridge, UK) and β-actin (1:1000; anti-mouse, cat. no. 4967S; Cell Signalling Technology, Milan, Italy) overnight at 4 °C. For histone protein extraction, we used Abcam histone extraction kit (AB113476, Abcam, Cambridge, UK) according to manufacturer’s protocol. For protein detection, rabbit primary H3K18Lac (1:1000; PTM-1406, PTM-biolabs, IL, USA) and H3 (1:1000; AB18521, Abcam, Cambridge, UK) were used. The next day, the membranes were washed three times in PBS for 5 min and incubated with secondary infrared anti-mouse IRDye800CW (1:5000) in PBS/0.5% Tween-20 for 1 h at room temperature. All antibodies were diluted in Odyssey Blocking Buffer. Protein bands intensity was quantified and normalized to β-actin levels [71 (link),72 (link),73 (link),74 (link)].

Ishikawa and KLE cells (3 × 10⁶) were seeded into 100-mm dishes, and after treatment with 1% formaldehyde, the cells were lysed with 1 ml of RIPA lysis buffer. Genomic DNA was isolated and sheared into 200–400-bp fragments using a sonicator. After centrifugation, the supernatants were collected, and the chromatin was precipitated with anti-H3K18la (1:50; PTM-1406, PTM Bio, China) or IgG (30000-0-AP, Proteintech, China) at 4 °C overnight. The following steps were conducted according to the manufacturer’s instructions for a ChIP assay kit (P2078, Beyotime, China). The primers designed for specific promoter regions of USP39 are listed in Supplementary Table 1.

Fresh tissues or adherent cells were washed twice with PBS and then lysed on ice for 1 h with RIPA lysis buffer (Servicebio, China) containing a cocktail and PMSF. After centrifugation, total protein concentrations were determined using a BCA protein assay kit (Biosharp, China). Individual samples (20 μg/lane) were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10–12% gels and transferred onto polyvinylidene difluoride membranes. After incubation with 5% dry milk in TBST at room temperature for 1 h, the membranes were washed and incubated with pan-anti-Kla (PTM-1401, PTM Bio, China), anti-H3K18la (PTM-1406, PTM Bio, China), anti-USP39 (23865-1-AP, Proteintech, China), anti-PGK1 (17811-1-AP, Proteintech, China), anti-PI3K (T40115, Abmart, China), anti-p-PI3K (T40116, Abmart, China), anti-AKT (T55561, Abmart, China), anti-p-AKT (T40067, Abmart, China), anti-HIF-1α (TA1009, Abmart, China), anti-β-Actin (AC038, ABclonal, China), and anti-H3 (17168-1-AP, Proteintech, China) at 4 °C overnight. After washing the membranes, the bound antibodies were detected with horseradish peroxidase-conjugated secondary antibodies and visualized using an enhanced chemiluminescence (ECL) kit (Biosharp, China). The relative expression or modification levels of the targets to those of β-Actin or H3 were determined by densitometric analysis using the ImageJ software.