Polyclonal rabbit anti gapdh antibody

Bacterial cells were suspended in 400 μl of Tris-buffered saline (TBS) with a protease inhibitor cocktail (Roche, Mannheim, Germany); the resulting suspension was transferred to a 2-ml screw-cap microcentrifuge tube containing 0.4 g of 0.2-mm glass beads. Tubes were subjected to 5 rounds of shaking with a bead crusher at 3,200 rpm for 1 min alternating with 1-min incubations on ice. To assess cell surface display of GAPDH, bacterial cells were centrifuged (8,000 × g, 4°C, 5 min) and the supernatant was decanted. The pellet then was subjected to 3 rounds of washing with PBS at pH 4.2 or pH 7.3, followed by centrifugation (20,000 × g, 4°C, 15 min). Following the final centrifugation, the pellets (corresponding to cell debris and insoluble material) and supernatants (corresponding to soluble released material) were analyzed separately by Western blotting using a primary antibody (polyclonal rabbit anti-GAPDH antibody, 1/5,000; GeneTex, CA, USA) and a secondary antibody (anti-rabbit IgG [whole molecule] peroxidase antibody, 1/5,000; Sigma-Aldrich, MO, USA). Labeling was detected using ImageQuant LAS 500 (GE Healthcare, WI, USA).

Aliquots (50 µg) of cell lysates were resolved using 10% acrylamide gels of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transblotted onto an Immobilon^TM-P membrane (Millipore, Bedford, MA, USA). After blocking with 5% skimmed milk, the membranes were incubated with various primary antibodies, and immunoreactivity was detected using horseradish-conjugated secondary antibody and visualized using an enhanced chemiluminescence kit (PerkinElmer, Boston, MA, USA). The following primary antibodies were used: mouse monoclonal anti-αSMA antibody (1:1000, Cat. No. sc-32251, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), rabbit monoclonal anti-Smad 2/3 (1:1000, Cat. No. 04-914, EMD Millipore Corporation, Temecula, CA, USA), rabbit monoclonal anti-Phospho-Smad2 antibody (1:1000, Cat. No. 04-935, EMD Millipore Corporation), mouse monoclonal anti-human MMP1 antibody (1:1000, Cat. No. MAB3307, EMD Millipore Corporation), mouse monoclonal anti-MMP2 antibody (1:1000, Cat. No. MAB3308, EMD Millipore Corporation), and rabbit polyclonal anti-GAPDH antibody (1:1000, GeneTex International Corporation, Hsinchu City, Taiwan).

Most of the chemicals were obtained from Sigma (St. Louis, MO, USA). Structurally defined, novel, and pure (>98.9% purity) garcinielliptone FC [7 (link)] and justicidin A [8 (link)] were gifts from Dr. Chun-Nan Lin (School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan) and formosanin C (FC) was kindly donated by Dr. Shen-Jeu Won (College of Medicine, National Cheng Kung University, Tainan, Taiwan) [9 (link)]. Sorafenib (Nexavar^®) was purchased from Bayer AG (Leverkusen, Germany). C11-BODIPY was obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Antibodies for Western blotting were purchased from the following vendors: rabbit monoclonal anti-CD71 (TFRC) and anti-ferritin heavy chain 1 (FTH1) antibodies, Cell Signaling Technology, Danvers, MA, USA; rabbit polyclonal anti-FPN/SLC40A1 antibody, Novus Biologicals, LLC, Littleton, CO, USA; rabbit polyclonal anti-ARA70 (NCOA4) antibody, Bethyl Laboratories, Inc., Montgomery, TX, USA; rabbit polyclonal anti-LC3B antibody, Abcam, Cambridge Science Park, Cambridge, UK; rabbit polyclonal anti-GAPDH antibody, GeneTex, Irvine, CA, USA; mouse monoclonal anti-β-actin antibody, Sigma; goat anti-rabbit (H+L) and goat anti-mouse IgM+IgG+IgA (H+L) HRP conjugate antibodies, Millipore Corp., Billerica, MA, USA.