Horseradish peroxidase hrp conjugated anti rabbit secondary antibody

The following reagents were used: Dulbecco’s Modified Eagle Medium (DMEM) (GE Healthcare Life Sciences, Logan, UT); Fetal Bovine Serum (FBS) (Atlanta Biologicals, Lawrenceville, GA); penicillin-streptomycin (Invitrogen, Carlsbad, CA); WST-1 proliferation assay kit (Roche, Indianapolis, IN); High-Capacity RNA-to-cDNA™ Kit and SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA); Diff-Quick cell staining kit (Dade Behring, Inc., Newark, DE); anti-human IL-8 ELISA Kit (R&D Systems Inc., Minneapolis, MN); Gemcitabine (Sigma-Aldrich, St. Louis MO). Antibodies used were: anti-CD45, -CD68, -F4/80, -TGF-β1, -IL-8 (Abcam, Cambridge, MA), anti-arginase-1, anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and anti-β-actin (Sigma-Aldrich). Western blotting SuperSignal West Femto Maximum sensitivity substrate kit was purchased from Thermo Scientific (Logan, UT). Immunohistochemical analysis reagent EZ-Dewax (Biogenex, Fremont, CA); background sniper, polymer, and probe were purchased from Biocare Medical (Concord, CA).

MCF-7 and MDA-MB-231 cells were obtained from ATCC (Manassas, VA). DMEM/F12, fetal bovine serum (FBS), Lipofectamine TM Reagent was purchased from Invitrogen (Paisley, UK). The anti pAkt, Akt, pmTOR, mTOR, GAPDH and horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). The anti Beclin 1, P62, ILC3, Caspase 3, Bcl2 and Bax were purchased from Proteintech (Wuhan, Hubei, China). Enhanced chemiluminescence (ECL) assay kit was purchased from Amersham (Louisville, Co).

Reagents used were as follows: letrozole (Sigma, L6545), 17β-estradiol (E2) (Sigma, 491187), celecoxib (Sigma, PZ0008), PGE2 (Sigma, P5640), tunicamycin (Sigma, T7765), salubrinal (Calbiochem, 324895), rapamycin (Sigma, R0395), arachidonic acid (Sigma, A9673), MTT (Sigma, M2128), Z-VAD-fmk (Sigma, V116), bafilomycin A1 (Sigma, B1793), 3-MA (Sigma, 08592), Hoechst 33342 (Sigma, B2261), acridine orange (Sigma, A6014), DMSO (Sigma, D2650). letrozole, E2, celecoxib, PGE2, tunicamycin, and salubrinal were dissolved in DMSO, while MTT, Hoechst 33342, and acridine orange were dissolved in phosphate-buffered saline (PBS).
Antibodies were obtained from the following sources: antibodies against Beclin 1, COX-2, EP-4, β-actin, Bcl-2, BAX, phospho-4EBP1 and phospho-Akt (S473) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); Phospho-p70-S6K(T389), eIF2α, phospho-eIF2α, Raptor, phospho-mTOR(S2448), caspase 3, and phospho-S6(S235/236) were from Cell Signaling Inc (Beverly, MA); LC3, ATG5, protein disulfide isomerase (PDI) were from Abcam (Cambridge, MA); horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody and horseradish peroxidase (HRP)-conjugated anti-mouse secondary antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Sporulation was induced by exhaustion by growing cells in DSM (Difco Sporulation Medium) as described elsewhere [24] . After a 30 hours of incubation at 37°C, spores were collected, washed four times, and purified as described by Nicholson and Setlow [29] using overnight incubation in H₂O at 4°C to lyse residual sporangial cells. Spore coat proteins were extracted from a suspension of spores by SDS-dithiothreitol (DTT) [24] , or NaOH [29] treatment as previously described. The concentration of extracted proteins was determined by using Bio-Rad DC protein assay kit (Bio-Rad), and 20 µg of total spore coat proteins were fractionated on 12,5% SDS polyacrylamide gels and electrotransferred to nitrocellulose filters (Bio-Rad) for Western blot analysis following standard procedures. CotH-, CotA-, CotC-, CotB- and CotG-specific antibodies were used at a working dilutions of 1∶150 for CotH detection and 1∶7000 for CotA, CotC, CotB and CotG detection. Then an horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody was used (Santa Cruz). Western blot filters were visualized by the SuperSignal West Pico chemiluminescence (Pierce) method as specified by the manufacturer.

Treated cell pellets were suspended in lysis buffer with a cocktail of protease inhibitors (Promega, USA). Protein detection by western blot was performed by electrophoretic transfer of equal amounts of SDS-PAGE separated proteins to polyvinyl difluoride (PVDF) membranes (Biorad, USA), incubated with the following primary antibodies: ZAP-70 #2705, LAT #4553, Phosphorylated-LAT #3584, SLP76 #4958 Phospho-SLP76 #92711, p-ZAP70 #2717, p38 MAPK #9212 (Cell Signalling Technology, USA) Actin (C-2; Santa Cruz Biotechnology Inc., USA) and GADPH (6C5; Santa Cruz Biotechnology Inc., USA). Either a horseradish peroxidase (HRP)-conjugated anti-rabbit secondary antibody (Santa Cruz Biotechnology Inc., USA) or an HRP-conjugated mouse IgG kappa binding protein (m-IgGκ BP) (Santa Cruz Biotechnology Inc., USA) was used for primary antibody detection via chemiluminescence (GE Healthcare, UK).