Ab236621

Cultured cells and GC tissues were lysed in cell lysis buffer supplemented with a protease inhibitor cocktail and phosphatase inhibitor cocktail. The protein concentration was measured using a BCA protein assay. The following primary antibodies were used: anti-4HNE monoclonal antibody (1 µg/ml; MHN-020P; Jaica), anti-SRF polyclonal antibody (1:1000; 16821-1-AP; Proteintech; RRID:AB_2194384), anti-AGPS antibody (1:1000; ab236621; Abcam; RRID:AB_2921211), anti-phospho-ERK1/2 (Thr202/Tyr204) polyclonal antibody (1:1000; 28733-1-AP; Proteintech; RRID:AB_2881202), anti-ERK1/2 polyclonal antibody (1:1000; 51068-1-AP; Proteintech; RRID:AB_2250380), anti-phospho-MEK1 (Ser298) monoclonal antibody (1:1000; 68047-1-Ig; Proteintech; RRID:AB_2918789), anti-MEK1/2 polyclonal antibody (1:1000; 11049-1-AP; Proteintech; RRID:AB_2140649), and anti-β-actin monoclonal antibody (1:1000; 66009-1-Ig; Proteintech; RRID: AB_2687938), and anti-AGPAT3 polyclonal antibody (1:1000; 25723-1-AP; Proteintech; RRID:AB_2880209). The following secondary antibodies were used: HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (1:5000; SA00001-2; Proteintech; RRID: AB_2722564) and HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (1:5000; SA00001-1; Proteintech; RRID: AB_2722565).

Immunohistochemical staining was performed using 5-μm-thick sections of 4% paraformaldehyde-fixed paraffin-embedded tissue samples. The following primary antibodies were used: anti-AGPAT3 antibody (1:200; DF3642; Affinity; RRID: AB_2836014), anti-4HNE monoclonal antibody (5 µg/ml; Jaica), and anti-AGPS antibody (1:700; ab236621; Abcam; RRID: AB_2921211). Goat anti-rabbit IgG and goat anti-mouse IgG were used as secondary antibodies. Immunohistochemical staining was performed using a Vector DAB kit. Notably, immunohistochemical staining was performed to examine the expression profiles of AGPS and AGPAT3 in gastric specimens as described previously; the expression profiles were evaluated and scored by two pathologists at Nanfang Hospital for the staining intensity (scale = 0–3) and staining frequency (scale = 0–4). For statistical analysis, the expression levels of the AGPS and AGPAT3 proteins were represented by an expression score ranging from 0 to 12 and calculated using the formula intensity×frequency.