Ab168379

The collected cell pellet was lysed with 1 × SDS buffer and centrifuged (13,000 rpm, 4 °C) for 10 min after sonication. After concentration detection, the extracted protein was separated by 8–12% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). Membranes were immunoblotted with anti-rabbit OLA1(HPA035790, 1:500, Sigma, USA), HK2 (2867S, 1:1000, CST, USA), PDHA1 (ab168379, 1:1000, Abcam, UK), m⁶A (ab151230, 1:1000, Abcam, UK), and anti-mouse GAPDH (1:2000, Zsbio, China) after sealed with 2% BSA, and the membranes were incubated with hybrid secondary antibody, the data was statistically collected by Fluor Chem V2.0 (Alpha Innotech Corp, USA).

After dewaxing and hydrating the FFPE, the EDTA technique was used to restore the tissue antigen. Tissue peroxidase was rendered inactive using hydrogen peroxide. The tissue was then immersed in primary antibody solution and kept in a wet box at 4° Celsius for 12 h. After that, the tissue sat at room temperature for half an hour to rewarm. After adding a second antibody labeled with horseradish peroxidase and letting it incubate for 60 min at room temperature, the tetramethylbenzidine color was seen. The favorable color reaction of the brownish-yellow particles was seen with an optical microscope.
This research made use of the following primary antibodies: CK (ab52625, Abcam), FDX1 (NBP1-89227, Novus), PDHA1 (ab168379, Abcam), PD-L1(ab205921, Abcam) and CD8 (14-0081-82, Invitrogen Antibodies). The breadth and number of tissues infiltrated by CD8⁺ T lymphocytes were used to establish immune classifications of GC [39 (link),40 (link)]. Tumor parenchyma and stroma were infiltrated by CD8⁺ T lymphocytes, a hallmark of the immune-inflamed subtype. CD8⁺ T cells infiltration was found exclusively in the peritumour stroma, but not in the parenchyma, marking the excluded immune subtype. The lack of CD8⁺ T cells in tumor parenchyma and stroma was a hallmark of the deserted immune subtype.

We lysed GC cells using the Radio Immunoprecipitation Assay buffer (BioRad). The lysate was separated via SDS‐PAGE, with the obtained proteins being transferred to polyvinylidene (PVDF) membranes (Millipore). We blocked membranes with 5% skimmed milk for 2 hours at 37°C, and then incubated at 4°C overnight with the following specific primary antibodies: PDK4 (1/1000, ab89295; Abcam), c‐E1α (1/1000, ab168379; Abcam), phospho‐Pyruvate Dehydrogenase (PDH)‐E1α (Serine 293; 1/1000, ab177461; Abcam), and GAPDH (1/1000, ab181602; Abcam). We washed the membranes with Tris Buffered Saline with Tween 20 (TBST), and incubated them with HRP‐conjugated AffiniPure goat anti‐rabbit (1/5000, SA00001‐2; Proteintech) or anti‐mouse IgG (H + L) (1/5000, SA00001‐1; Proteintech) at 37°C for 2 hours. The PVDF membranes were washed three times with TBST buffer and then observed with an electrochemiluminescence (ECL) detection system. As an internal control, we selected GAPDH.