Horseradish peroxidase conjugated secondary antibody

HEK293T cells transfected with pTriEx-mPB and pTriEx-NP-mPB were washed twice with ice-cold PBS and lysed in lysis buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 10 µg/ml aprotinin, and 50 µM ρ-APMSF). The lysates were centrifuged, and the protein extracts were separated on 8% SDS-PAGE and transferred to Immobilon transfer membranes (PVDF) (Millipore, Bedford, MA, USA). The membranes were washed, incubated in TBST containing 5% skim milk for 1 h, and treated overnight with an anti-His tag (1∶2000; GeneTex, Inc., Irvine, CA, USA), anti-GFP (1∶5000; GeneTex, Inc.), or anti-α-tubulin antibody (1∶5000; Abcam plc, Cambridge, UK) at 4°C. A horseradish peroxidase-conjugated secondary antibody (1∶5000; GeneTex, Inc.) was applied to allow color reaction for detection. For nucleo-cytoplasmic separation experiments, cytoplasmic and nuclear protein fractions were extracted from transfected HEK293T cells with the ProteoJET cytoplasmic and nuclear protein extraction kit (Thermo Fisher Scientific Inc.).

Samples of the left hippocampus or cortex were homogenized in PBS containing complete protease inhibitors in a glass vesicle; the resulting homogenate was centrifuged at 12,000 rpm at 4°C for 20 min; and the protein concentrations of the supernatant thus obtained determined with the BCA protein assay kit. Proteins (30 μg) were subsequently separated by 8-10% SDS-PAGE and then blotted onto polyvinylidene difluoride (PVDF) membranes with a transfer unit (Bio-Rad Inc.). For the relative quantification of the proteins, these membranes were thereafter incubated with antibodies against α7 (1:1000), α4 (1:1500) and β2 (1:1000), cleaved caspase-3 (1:1000, Gene Tex, USA), Bcl-2 (1:1000, CST, USA), Bax (1:1000, CST, USA), Snap-25 (1:1000, Gene Tex, USA), Syn (1:1000, Gene Tex, USA) or β-actin antibody (1:5000, Gene Tex, USA) at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Gene Tex, USA) for 60 min. Finally, these membranes were incubated in ECL Plus (Pierce, Thermo Fisher Scientific) reagent and the signals thus obtained visualized by exposure to hyper-performance chemiluminescence film for 30 sec to 5 min.

After lysis, lung tissue samples (50 μg) were run on 12.5% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blocking with 5% bovine serum albumin in Tris-buffered saline, the membranes were incubated overnight at 4 °C with primary antibodies, anti-MyD88 (Gene Tex, GTX112987), or anti-phospho-NFκB (Gene Tex, GTX50254). Then, the membranes reacted with horseradish peroxidase-conjugated secondary antibody (Gene Tex, GTX213110-01) at room temperature for 1 h. As a loading control, the same blots were re-probed with anti-GAPDH (Gene Tex, GTX100118) and anti-mouse horseradish peroxidase antibodies. All samples were performed in triplicate.

Cells were lysed with lysis buffer (Cell Signaling Technology) of cold phosphate-buffered saline (PBS). The supernatants of the cell lysate were centrifuged at 14,000 rpm for 10 min at 4 °C. The bicinchoninic acid protein assay (Thermo Scientific) was used to detect the total protein concentrations. Equal amounts of proteins obtained from each group and mixed with loading buffer (Biotools, Taipei, Taiwan) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and were then transferred onto polyvinylidene fluoride (PVDF) membranes (Merck Millipore). After blocking with 5% bovine serum albumin (BSA) in PBS, the membrane was incubated with indicated antibodies in PBS at 4 °C overnight. After being washed twice with TBST (tris buffered saline containing 0.05% Tween 20), the membrane was incubated with horseradish-peroxidase-conjugated secondary antibodies (GeneTex) at room temperature for 2 h. The antibody-labeled membrane was washed twice with TBST. Then, enhanced chemiluminescence (ECL) substrates (Merck Millipore and Biotools) were used for detection in an ImageQuant LAS 4000 (GE Healthcare, Chicago, IL, USA) system [15 (link)]. Furthermore, the expression levels of target proteins were quantified by Image J software (version 1.52q, NIH, Bethesda, MD, USA) with normalizing to each internal control.

Antibodies against AXL, pIGF-1R (Y1135/6), IGF-1R, pSrc (Y416), Src, pMet (Y1234/5), Met, tubulin, and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against cleaved PARP were purchased from BD Biosciences (San Jose, CA, USA). Antibodies against pAXL (Y702) were purchased from R&D systems (Minneapolis, MN, USA). Primary antibodies against IGF-1R and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against pIR/IGF-1R (Y1162/3) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Horseradish peroxidase-conjugated secondary antibodies were purchased from GeneTex (Irvine, CA, USA). Linsitinib, dasatinib, and bemcentinib (R428) were purchased from Selleckchem (Houston, TX, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. The detailed information on used primary and secondary antibodies, including vendor, catalogue number, application, and dilution ratio (or concentration) is listed in Table S1.