Ptm 1401

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.

T. gondii lysates (20 μg) were separated using SDS–PAGE, and the proteins were transferred to a polyvinylidene fluoride membrane (Catalog No. 162-0177, Bio-Rad, Hercules, CA). The membrane was blocked using 5% skim milk at 37 °C for 45 min and then incubated overnight at 4 °C with an anti-lactyllysine antibody (Catalog No. PTM-1401, PTM BIO). The membrane was washed four times with 1× PBS buffer and incubated with a secondary antibody at 37 °C for 50 min (Catalog No. 31430, Thermo Fisher Scientific). The procedure for the Western blotting assays of the recombinant proteins was the same as above (with an antibody dilution ratio of 1:200). Anti-TATA-binding protein and anti-β tubulin antibodies were purchased from PTM BIO (Catalog Nos. PTM-5007 and PTM-5028, PTM BIO).

Samples were embedded in paraffin and sliced into sections at a thickness of 4 μm. The sections were then incubated with anti-L-lactyl lysine rabbit mAb (pan-anti-Kla; PTM-1401, PTM Bio, China), anti-lactyl-histone H3 (Lys18) rabbit mAb (anti-H3K18la; PTM-1406, PTM Bio, China), and anti-USP39 (23865-1-AP, Proteintech, China) at 4 °C overnight. The slides were then rinsed three times with PBS, incubated with a secondary antibody at room temperature for 30 min in the darkness, and visualized after being stained with a DAB solution. Three randomly selected fields were observed under a microscope (Motic, China). The IHC staining scores, based on the staining intensity (SI) and the percentage of immunoreactive cells (PR), were evaluated by two independent observers who were blinded to the patient’s identity. The SI scores were assigned from 0 to 3 as follows: 0 = no staining; 1 = weak staining; 2 = moderate staining; and 3 = strong staining. The PR was scored from 1 to 4 as follows: 1 = 0–25%; 2 = 26–50%; 3 = 51–75%; and 4 = 76–100%. The PR and SI scores were multiplied to produce a weighted score for each patient. A score of 8–12 was defined as a high expression level, and a score of 0–7 was defined as a low expression.