Anti glyceraldehyde 3 phosphate dehydrogenase gapdh

PTNs were a generous gift from Won-Kyo Jung, Pukyong National University, Busan, South Korea. PTNs were prepared from Ecklonia cava collected along the Jeju Island coast of Korea as previously described [43 (link)]. Composition and chemical structures of PTNs in the ethanolic extracts were previously characterized [44 (link),45 (link)]. Easyef^TM, a commercial spray-type ointment containing rhEGF, was purchased from Daewong Pharmaceuticals (Seoul, South Korea). The primary antibodies used were as follows: anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5174), anti-p65-NF-κB (#8242), anti-IL-1β (#12242), and anti-HO-1(heme oxygenase) (#5853) purchased from Cell Signaling Technology (Danvers, MA, USA); anti-ASC (SC514414) and anti-NRF2 (SC1722) purchased from Santa Cruz Biotechnology (Dallas, TX, USA); anti-AQP3 (AB3276) purchased from Merck Millipore (Burlington, MA, USA); and anti-COX2 (#610203) purchased from BD Biosciences (Franklin Lakes, NJ, USA). Horseradish peroxidase (HRP)-conjugated secondary anti-rabbit and anti-mouse antibodies were purchased from Cell Signaling Technology.

Protein samples extracted from kidney tissues were loaded onto gradient polyacrylamide gels and then transferred onto nitrocellulose membranes. The membranes were probed with specific primary antibodies as follows: anti-NGAL (Santa Cruz Biotechnology), anti-cleaved caspase-3 (Cell Signaling, Danvers, MA, USA), anti-cleaved poly(ADP-ribose) polymerase-1 (PARP-1; Cell Signaling), anti-p53 (Cell Signaling), anti-Bax (Santa Cruz Biotechnology), anti-tumor necrosis factor-α (TNF-α; Abcam), anti-interleukin-6 (IL-6; Abcam), anti-nuclear factor-κB (NF-κB) p65 (Cell Signaling), anti-p-NF-κB p65 (Cell Signaling), anti-α-SMA (Sigma-Aldrich), anti-fibronectin (Abcam), anti-transforming growth factor-β (TGF-β; R&D Systems, Minneapolis, MN, USA), anti-p-Smad2/3 (Cell Signaling), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Cell Signaling) antibody. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence detection system (Thermo Fisher Scientific, Waltham, MA, USA) and analyzed using the ChemiDoc™ XRS+ Imaging System (Bio-Rad Laboratories, Hercules, CA, USA). The protein expression levels were normalized against GAPDH.

For Western blot analysis, the following antibodies were used: anti-LC3B (2775; Cell Signaling Technology), anti-SQSTM1/P62 (5114; Cell Signaling Technology), anti-ATG5 (CY5766; Abways), anti-fibronectin (ab32419; Abcam), anti-integrin α5 (CY5979; Abways), anti-integrin β1 (CY5469; Abways), anti-phospho-mTOR (Ser2448) (AF3308; Affinity Biosciences), anti-mTOR (AF6308; Affinity Biosciences), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (5174; Cell Signaling Technology). Horseradish peroxidase-labeled goat anti-rabbit (ASS1009; Abgent) secondary antibodies were used. The transfection reagents were Lipofectamine 2000 (11688-019; Invitrogen) and Lipo6000™(Beyotime). The immunoprecipitation (IP) reagent was included in the Pierce classic magnetic IP/co-IP kit (88804; Thermo Scientific).

Caco-2 cells were purchased from European Collection of Cell Cultures (ECACC, Public Health England Porton Down, Salisbury, UK). Cell medium, chemicals and reagents used for cell culture, and TcdA were purchased from Sigma–Aldrich (St. Louis, MO, USA), unless otherwise stated. Instruments, reagents, and materials used for western blot analysis were obtained from Bio-Rad Laboratories (Milan, Italy). Rabbit anti-zona occludens-1 (ZO-1), anti-occludin and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were procured from Cell Signaling Technology (Danvers, MA, USA). Rabbit anti-toll-like receptor 4 (TLR4), mouse anti-ZO-1, anti-Bcl-2-associated X protein (Bax), mouse anti-MyD88, rabbit anti-transforming growth factor-β-activated kinase-1 (pTAK1), and mouse anti-TAK1 antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and horseradish peroxidase (HRP) was obtained from Dako (Milan, Italy). Fluorescein isothiocyanate-conjugated anti-rabbit antibody and Texas red conjugated anti-mouse antibody were purchased from Abcam (Cambridge, UK), and custom oligonucleotides for electrophoretic mobility shift assay (EMSA) analysis were synthesized by TIB Molbiol (Berlin, Germany).