Rabbit igg isotype control

Macrophages were stimulated with or without H. capsulatum (MOI = 5) for 1 h before lysis with non-denaturing lysis buffer [20 mM Tris HCl pH 8, 137 mM NaCl, 1% Nonidet P-40 (NP-40), 2 mM EDTA] supplemented with protease inhibitor cocktail (Sigma-Aldrich) and 25 mM N-ethylmaleimide (NEM). Whole cell lysates were incubated with antibodies against NLRX1 (Cell signaling), TUFM (Abcam), or rabbit IgG isotype control (GeneTex) at 4°C overnight followed by mixing with Protein A agarose beads (Merck Millipore) at 4°C for another 4 h. Lysate beads mixture was washed with IP washing buffer (0.1% NP-40 in PBS). The immunoprecipitates were eluted by being boiled in PhosphoSafe Extraction Reagent cell lysis buffer (Novagen) containing 1 × SDS-PAGE Loading Buffer (BIOTOOLS) and subjected to Western blotting.

The CytoFLEX-S was used for the detection of (PE) IRE1α and (PerCP) pIRE1α in the cell lines. For the characterization of M1/M2 macrophages the surface and intracellular markers, referenced in the M1/M2 macrophage polarization section, were used and analyzed in the BD Bioscience FACS Calibur. Compensation was done by fluorescence minus one (FMO) for all the antibodies. The pIRE1α (GeneTex) and its Rabbit IgG isotype control (GeneTex) antibodies were conjugated using the LYNX Rapid PerCP Antibody Conjugation Kit, as per the manufacture's recommendations.

Syk inhibitors SykI and BAY 61-3606, JNK inhibitor SP600125, ERK inhibitor U0126, and p38 inhibitor SB203580 were obtained from Calbiochem-Merck. Laminarin and zymosan were purchased from InvivoGen. Peptide inhibitor for NADPH oxidase assembly, gp91ds-tat, was purchased from AnaSpec. Diphenyleneiodonium chloride (DPI) and Mito-TEMPO were obtained from Sigma-Aldrich. LysoTracker Red was obtained from Life Technologies.
Antibodies against LC3, Rubicon, NLRX1, phospho (p)-p40-phox (Thr154), p-JNK (Thr183/Tyr185), p-ERK1/2 (Thr202/Tyr204), p-p38 (Thr180/Tyr182), p-c-Fos (Ser32), p-IKKα/β (Ser176/180), p-IκBα (Ser32), IκBα, and p-NFκBp65 (Ser536) were purchased from Cell Signaling. Antibodies against p-Syk (Tyr525), p-c-Jun (Ser63), and TUFM were obtained from Abcam. Anti-Syk, anti-ATG5, anti-β-actin, HRP-conjugated anti-rabbit IgG, and Rabbit IgG isotype control were purchased from GeneTex Inc. Receptor blocking antibodies against Dectin-1 (clone 2A11), Dectin-2 (clone D2.11E4), and CR3 (clone 5C6) were purchased from Bio-Rad (formerly AbD Serotec) and TLR2 (clone 6C2) was from eBioscience.