Rabbit anti human gapdh

Total protein was extracted from BT-20 and MDA-MB-231 cells using RIPA assay buffer (Thermo Fisher Scientific, Inc.), after which total protein was quantified using a bicinchoninic acid assay. Protein (30 µg) was loaded onto 12% SDS-PAGE gel and transferred to PVDF membranes. Non-fat milk (5%) was used for blocking at room temperature for 2 h. The following primary antibodies were then incubated at 4°C for 18 h: Rabbit anti-human GLUT1 (1:1,500; cat. no. ab15309; Abcam) and rabbit anti-human GAPDH (1:1,300; cat. no. ab8245; Abcam). Horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (1:1,000; cat. no. MBS435036; MyBioSource, Inc.) were subsequently used incubated at 24°C for 2 h. A Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was used to produce signals and ImageJ v1.48 software (National Institutes of Health) was used to process data.

Total protein extraction from HT-1376 and HT-1197 cell lines was performed using a radioimmunoprecipitation assay lysis solution (Thermo Fisher Scientific, Inc.), and BCA assay was used for protein quantification. Subsequently, 20 µg protein from each sample was subjected to 10% SDS-PAGE gel electrophoresis, followed by gel transfer to polyvinylidene fluoride membranes. Membranes were blocked with 5% skimmed milk at room temperature for 2 h, followed by washing with PBS and incubation with primary antibodies including rabbit anti-human EGFR (1:2,000; cat. no. ab131498; Abcam, Cambridge, UK) and rabbit anti-human GAPDH (1:1,000; cat. no. ab8245; Abcam) overnight at 4°C. Following washing with PBS, the membranes were incubated with an anti-rabbit IgG-horseradish peroxidase secondary antibody (1:1,000; cat. no. MBS435036; MyBioSource, Inc., San Diego, CA, USA) at room temperature for 1 h. Then, enhanced chemiluminescent reagents (Sigma-Aldrich; Merck KGaA) were added to detect the signals. Membranes were scanned using MYECL™ Imager (Thermo Fisher Scientific, Inc.), and ImageJ v1.46 software (National Institutes of Health, Bethesda, MD, USA) was used to normalize the relative expression level of EGFR to the endogenous control GAPDH.

Protein lysates of HEK-293T cells were extracted with RIPA buffer (PIERCE) and protease inhibitor cocktail (Roche). Loading buffer (Life Technologies) was added, and samples were heated to 95°C for 5 minutes and subjected to electrophoresis in 12% NuPAGE Bis-Tris gels (Life Technologies). Gels were transferred to nitrocellulose membranes (Life Technologies) and probed with the primary antibodies rabbit anti-V5 (Bethyl Laboratories), mouse anti-TMPRSS2 (Santa Cruz), and rabbit anti-human GAPDH (ABCAM) and subsequently with the secondary antibody IRDye 800RD goat anti-rabbit and IRDye 680RD goat anti-mouse (LI-COR Biosciences). Membranes were visualized and analyzed using the Odyssey CLx system (LI-COR Biosciences). For TMPRSS2 quantity estimations, recombinant TMPRSS2 was subjected to gel electrophoresis with protein lysates from TMPRSS2-overexpressing cells.

U251 cells were lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.), and the protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein (40 µg/lane) was separated via 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.). Following this, the membrane was blocked using 5% non-fat milk in PBS (Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA) at room temperature for 3 h. Subsequently, the PVDF membrane was incubated with rabbit anti-human polyclonal KDM2A (1:1,000; cat. no. ab191387; Abcam, Cambridge, MA, USA) and rabbit anti-human GAPDH (1:1,000; cat. no. ab9485; Abcam) primary antibodies at room temperature for 2 h. Following washing with PBS for 10 min, the PVDF membrane was incubated with horseradish peroxidase-tagged goat anti-rabbit secondary antibodies (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. Membranes were then washed with PBS for 10 min, and the protein bands were visualized using an Enhanced Chemiluminescence Western Blotting kit (Pierce; Thermo Fisher Scientific, Inc.), in accordance with the manufacturer's protocol. Protein densitometry was performed using ImageJ software (version 1.41; National Institutes of Health, Bethesda, MD, USA).

SNU-5 cells were lysed in cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc.), and the protein concentration was determined using a Bicinchoninic Acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.). Protein was separated by 10% SDS-PAGE and then transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific, Inc.), which was then blocked in 5% non-fat milk in PBS (Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich; Merck) at room temperature for 3 h. Subsequently, the PVDF membrane was incubated with rabbit anti-human polyclonal KLF5 (1:50; cat. no. ab137676; Abcam, Cambridge, MA, USA) or rabbit anti-human GAPDH (1:100; cat. no. ab9485; Abcam) primary antibodies at room temperature for 3 h. Following washing with PBS for 10 min, the PVDF membrane was incubated with a goat anti-rabbit secondary antibody (1:5,000; cat. no. ab7090; Abcam) at room temperature for 1 h. After further washing with PBS for 10 min, the protein bands were detected using an Enhanced Chemiluminescence Western Blotting kit (Pierce; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocols, and then quantified using Image Lab analysis software version 3.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA).