Glyceraldehyde 3 phosphate dehydrogenase gapdh antibody

PGF2α was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). The ELISA kits for mouse IL-6 and mouse VEGF were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Phospho-specific p44/p42 MAP kinase antibodies, p44/p42 MAP kinase antibodies, phospho-specific p38 MAP kinase antibodies, and p38 MAP kinase antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Buckinghamshire, UK). Negative control-small interfering RNA (siRNA) (Silencer Negative Control #1 siRNA (Neg)) and HSP22-siRNA (s206904 (#1) and s96094 (#2)) were purchased from Ambion (Austin, TX, USA). Other materials and chemicals were obtained from commercial sources.

TGF-β was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Onalespib was purchased from Selleckchem (Houston, TX, USA). Geldanamycin was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). 17-DMAG and SP600125 were purchased from Calbiochem-Novabiochem Co. (La Jolla, CA, USA). SB431542 was purchased from Funakoshi Co., Ltd. (Tokyo, Japan). HSP27 antibodies, phosphorylated HSP27 antibodies, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Phospho-specific Smad2, Smad2, phospho-specific p44/p42 MAPK, p44/p42 MAPK, phospho-specific p38 MAPK antibodies, p38 MAPK antibodies, phospho-specific SAPK/JNK antibodies, and SAPK/JNK antibodies, were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). An ECL Western blotting detection system was obtained from GE Healthcare Life Sciences (Chalfont, UK). Other materials and chemicals were obtained from commercial sources. Onalespib, Geldanamycin, 17-DMAG, SP600125, and SB431542 were dissolved in dimethyl sulfoxide (DMSO). The maximum concentration of DMSO was 0.1%, which did not affect the assay for Western blot analysis [21 (link)–23 (link)].

Total proteins were subjected to Western blot analysis [60 (link)]. Blots were incubated overnight at 4 °C with antibodies against cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4), bax, bcl2, and parp1 (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and immunoreactive bands were visualized with the ECL Western blotting detection system (Amersham Pharmacia Biotech). To ensure equal loading of proteins, membranes were stripped and incubated overnight with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology).

Fifty μg of protein was subjected to western blot analysis [62 (link)]. Blots were incubated overnight at 4°C with antibodies against GPER, Cyclin E (CCNE), Cyclin B1 (CCNB1), phospho-Rb, Cytochrome c, Bax, Bcl-2, Parp1, pERK1/2-ERK2 (all from Santa Cruz Biotechnology, Santa Cruz CA, USA). Membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ) and immunoreactive bands were visualized with the ECL western blotting detection system (Amersham Pharmacia Biotech, Piscataway, NJ). To assure equal loading of proteins, membranes were stripped and incubated overnight with Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology).

Recombinant murine IL-6 was purchased from CellSystems (Troisdorf, Germany), TNF-α from Miltenyi Biotec (Bergisch Gladbach, Germany), LCN2 from Enzo Life Sciences (Lörrach, Germany), macrophage colony-stimulating factor (M-CSF) and platelet-derived growth factor (PDGF) from R&D-Systems (Minneapolis, MN), and human oxLDL from Source Bioscience (Nottingham, UK). F4/80-PE antibody was obtained from eBioscience (San Diego, CA), appropriate immune globulin (Ig)G-PE as well as the biotinylated CD31c antibody was obtained from BD Bioscience (San Jose, CA). LCN2 antibody was purchased from Abcam (Cambridge, UK), MOMA2 antibody was obtained from Acris (Herford, Germany), and iNOS antibody was purchased from Life Technologies (Darmstadt, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), arginase (Arg)1 and chitinase 3-like 3 (Ym1) antibody were from R&D Systems. Biotinylated secondary antibody and ImmPRESS HRP Polymer Detection Kit (anti-rat IgG and anti-rabbit) were obtained from Vector Laboratories (Burlingame, USA). Appropriate alkaline phosphatase (AP)-conjugated secondary antibody was purchased from Sigma Aldrich (Seelze, Germany). Appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies were from GE Healthcare (Munich, Germany).