Df3645

The extraction of overall proteins of tissues and cells was made with FLAG lysis buffer with a protease inhibitor cocktail. A BCA kit was used to measure protein concentrations. Proteins were electrophoresis, transferred, and blocked, then incubated with the diluted primary antibodies at 4 °C overnight. PSMD3 (Affinity biosciences, DF3645, 1:1000), ILF3 (Abcam, ab92355, 1:1000), ERBB3 (Affinity biosciences, DF6144, 1:1000), p-EGFR (Abcam, ab32430, 1:1000), EGFR (Abcam, ab32077, 1:1000), and GAPDH (Cell signaling, 21185, 1:1000) were all used in this study. Horseradish peroxidase-labeled secondary antibodies was adopted to incubate the membranes after extensive washes, and a strengthened chemiluminescent kit with a ChemiDoc XRS testing system was adopted to test signals. ImageJ software was adopted to analyze the signals.

The overnight incubation of tissue sections from clinical patients or xenografts was made with anti-PSMD3 antibody (Affinity biosciences, DF3645, 1:100), anti-ILF3 (Abcam, ab92355, 1:100), and anti-Ki67 at 4 °C. 0.3% H₂O₂ was used for blocking endogenous peroxidases for 15 min. After three times of section washing, streptavidin and biotin-conjugated HRP was put into the tissues for incubation for 1 h at 25℃ and then stained with 3’-diaminobenzidine for 30 min and stopped by 5-minute rinsing in H₂O. IHC staining results were imaged with a Leica upright microscope.
Two experienced pathologists conducted an independent assessment of immunohistochemical outcomes, which were then scored based on Friedrich’s standard. The ratio of positive tumor cells was assessed with a mark of 0 (0–5%), 1 (6–20%), 2 (21–50%), or 3 (51–100%). The marks of the staining intensity and the ratio of positive cells were multiplied to obtain the final score. A final score ≤ 1 indicated low expression, while a score > 1 was defined as a high expression.