Ab203398

THP-1 cells were rinsed twice with ice-cold PBS and lysed with RIPA buffer (150 mM NaCl, 1 mM EGTA, 50 mM Tris pH 7.4, 10% glycerol, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and protease inhibitor cocktail). The protein concentration of the cell lysates was measured using the Bradford reagent (Thermo Fisher, USA). Next, 30 μg of each protein sample was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) paper. The resultant NC papers were then incubated overnight at 4 °C with a range of specific primary antibodies, including signal transducer and activator of transcription (STAT6) (1:1000, ab32520, Abcam, USA), p-STAT6 (1:1000, ab28829, Abcam, USA), peroxisome proliferator-activated receptor γ (PPARγ) (1:1000, #2435, Cell Signaling, USA), high mobility group protein B1 (HMGB1) (1:5000, ab18256, Abcam, USA), and interleukin-4 receptor (IL-4 R) (1:1000, ab203398, Abcam, USA). After incubation with the indicated primary antibody, the proteins of interest were then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Millipore, USA) and visualized by the ECL detection system (Millipore, USA).

The immunohistochemical staining (IHC) helped detect the Rab1A/IL4Ra expression in 115 GC and para-cancer tissues. These GC tissues were fixed in formalin, embedded in paraffin, cut into 5um, and stained using IHC, based on our previous study ^{32 (link)}. Sections were incubated for two hours using the anti-Rab1A and anti-IL4Ra at 1:100 dilution at room temperature. The process was visualized using the tissue staining kit (Zhongshan Biotechnology, Beijing, China). The staining score was calculated based on our previous study. Five regions were randomly selected for staining evaluation, and the IHC score was determined by multiplying the staining intensity (0, negative; 1, weak; 2, moderate; and 3, strong) and extent (0, 0–5%; 1, 6–25%; 2, 26–50%; 3, 51–75%; and 4, > 75%). We considered 0 as − ,1 ~ 4 as + , 5 ~ 8 as ++, and 9 ~ 12 as +++, for the final staining score. Our study regarded ++ or +++ as a high expression and – or + as a low expression. We used Anti-Rab1A (1:100, Abcam, ab302545) and anti-IL-4Rα (1:100, Abcam, ab203398) antibodies for immunohistochemistry.