Anti rabbit igg hrp conjugate

Protease inhibitor and Phosphate inhibitor cocktails, 2-Deoxy-D-glucose, Oligomycin A, Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, Antimycin A and Rotenone were purchased from Sigma-Aldrich. HTH-01-015, a potent and selective NUAK1 inhibitor (23 (link)) was from Tocris (Bristol, UK). Fluorophores Tetramethylrhodamine Ethyl Ester, Perchlorate (TMRE), MitoTracker™ Green FM, and Hoechst 33,342 were from ThermoFisher. AccuRuler RGB plus protein ladder was purchased from MaestroGen Inc. (Hsinchu City, Taiwan). Anti-NUAK1 antibody (#4458) was from Cell Signaling (Danvers, MA, USA), and the anti-FLAG (M2) was from Sigma-Aldrich. Antibodies against β-Actin (AC-15), ATP5B (E-1), and TOM20 (F-10) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Total OXPHOS Rodent WB Antibody Cocktail (ab110413) was from Abcam (Cambridge, United Kingdom). Goat Secondary antibodies anti-mouse IgG-HRP and anti-rabbit IgG-HRP conjugates were purchased from Bio-Rad (Hercules, CA, USA). The anti-mouse Alexa-488 antibody (A11001) was from ThermoFisher.

The CC domain of Pikh-1 was inserted into the pF3A WG vector fused to a FLAG tag construct (Promega), while the Pikh-2_CC, and intact and truncated fragments of AvrPik-h were inserted into the pGEX-6P-1 vector to form a GST-fusion construct (GE Healthcare). The GST-fused proteins expressed in E. coli strain BL21 were used to pull down the FLAG-fused proteins synthesized in a wheat germ-derived in vitro transcription and translation system (Promega) using a MagneGST pull-down system (Promega), following the manufacturer's protocol. The proteins eluted from the beads were separated through a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-Rad). Protein blots were blocked with 3% w/v skimmed milk and then probed with either an anti-GST or an anti-FLAG monoclonal antibody (Sigma). Either goat anti-mouse (Sigma) or anti-rabbit IgG HRP conjugates (Bio-Rad) was used as the secondary antibody for subsequent detection via enhanced chemiluminescence (GE Healthcare). As an additional recognition specificity assay, two sets of the five Pik alleles, three versions of AvrPik-h alleles, as well as the Pikh-2_CC (the Pik-2 alleles share an identical CC domain) were tested by the above procedure.

Whole-cell protein lysates were prepared using RIPA buffer (iNtRON Biotechnology, Seongnam, Korea) supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland), and total protein samples were quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). After separation of equal amounts of the protein lysates on 10% Bis–Tris protein gels (Thermo Fisher Scientific, Waltham, MA, USA), transfer to PVDF membranes (Merck Millipore, USA), and blocking with 5% skim milk, the membranes were incubated with HRP-conjugated anti-β-actin, anti-phospho-ERK1/2, anti-total-ERK1/2, anti-phospho-AKT (S473), or anti-total-AKT antibodies (Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were washed in 0.05% Tween 20 in Tris-buffered saline and incubated with a 1:5000 dilution of anti-rabbit IgG -HRP conjugate (Bio-Rad, USA) as the secondary antibody. Specific bands were detected using a WEST-ZOL plus Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea).