Phosphosafe lysis buffer

BMDCs (2 × 10⁶) were treated with or without blocking antibody for 60 min before the addition of live or heat-killed H. capsulatum. For inflammasome analysis, cells were cultured in medium containing 0.1% heat-inactivated FBS and for signaling molecule analysis, in medium containing 10% FBS. Cells were detached from the wells and lysed with PhosphoSafe lysis buffer (MERCK) at different time points. Harvested cell-free supernatants were concentrated by 10-fold with Vivaspin 500 (GE Healthcare). Cell lysates were subjected to electrophoresis at 10% (for cell lysates) or 12.5% (for supernatants) SDS polyacrylamide gel and transferred to a 0.45 (for cell lysates) or 0.22 (for supernatants) μm PVDF membrane. The membrane was blocked with 5% non-fat milk and left in buffer containing anti-IL-1β p17 (R&D system), anti-Caspase-1 p20 (Adipogen), anti-NLRP3 (Adipogen), anti-ASC (Adipogen), anti-p-Syk (Abcam), anti-p-ERK (Cell Signaling), anti-p-JNK (Cell Signaling), anti-p-p38 (Cell Signaling) or anti-β-actin (GeneTex) antibody at 4°C overnight. The membrane was washed with TBST before addition of HRP-conjugated anti-goat IgG (1:3000), anti-rabbit (1:20,000) or anti-rat IgG (1:20,000). ECL reagent (PerkinElmer Life Science, Merck Millipore and GE Healthcare) was used for detection.

Cell lysis and immoblotting was essentially performed as described previously [22 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with 1× PhosphoSafe lysis buffer (Merck Millipore). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by polyacrylamide gel electrophoresis on mini-PROTEAN TGX any-kD precast gels, or TGX Stain-Free FastCast gels (BioRad) and blotted to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies either for 2 h at RT or overnight at 4 °C. After washing and incubation with HRP-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS+ System with Image Lab Software (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to either the total amount of protein in the same lane (when using the TGX Stain-Free FastCast gels), or to bands for the housekeeping gene GAPDH.

Cell lysis and immoblotting was essentially performed as described previously [18 (link)]. In brief, cells were washed once with ice-cold PBS and lysed with 1× PhosphoSafe lysis buffer (Merck Millipore). Following clearance of the lysates by centrifugation, their total protein concentrations were determined with the DC Protein Assay (BioRad). Equal amounts of proteins were fractionated by polyacrylamide gel electrophoresis on mini-PROTEAN TGX any-kD precast gels (BioRad) and blotted to PVDF membranes. Membranes were blocked with nonfat dry milk or bovine serum albumin and incubated with primary antibodies either for 2 h at RT or overnight at 4 °C. After washing and incubation with HRP-linked secondary antibodies, chemoluminescent detection of proteins was done on a ChemiDoc XRS imaging system (BioRad) with Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany). The signals for the proteins of interest were normalized to those for GAPDH or HSP90 (identity verified by size determination using the SM1841 molecular weight marker from Fermentas/Thermo Fisher Scientific (Supplementary Figure S7)).

RNA was extracted using RNeasy Mini kit (Qiagen) and quantified using the Nanodrop. qRT-PCR was performed using inventoried TaqMan Gene Expression Assays (Applied Biosystems). RNA expression values were normalised to GAPDH and all experiments were performed in triplicate. Cell lysates were harvested using PhosphoSafe lysis buffer (Merck Millipore Ltd, Feltham, UK) and Western blotting carried out as previously described [39 (link)]. Primary antibodies used were p ALK (Tyr-1604 and Tyr-1282/83), ALK, p ERK, ERK, pAKT, AKT, pSTAT3 (Cell Signaling, Leiden, The Netherlands), STAT3 (R&D Systems) MYCN and GAPDH (Santa Cruz Biotechnology, Heidelberg, Germany), all at 1:1000. Flow cytometry and Caspase 3/7 assays were performed as previously described [39 (link)].

Confluent cells were washed once with ice-cold PBS and lysed with 1 x PhosphoSafe lysis buffer (Merck Millipore). Following sonication and clearing, the total protein concentration of the supernatants was determined with the BioRad DC Protein Assay. Samples were subjected to gel electrophoresis using BioRad mini-PROTEAN TGX any-kD precast gels and blotted to 0.45 μm PVDF membranes. Membranes were blocked with non-fat dry milk or BSA and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare, Munich, Germany) were used for chemoluminescent detection of proteins on a BioRad ChemiDoc XRS imaging system.

Confluent cells were washed once with ice-cold PBS and lysed with 1 × PhosphoSafe lysis buffer (Merck Millipore). Cell lysates were sonicated and centrifuged for 10 min at 14,000× g and 4 °C following determination of total protein concentration in supernatants using BioRad DC protein assay. Samples containing equal amounts of protein were prepared using 3× SDS sample buffer and 125 mM dithiothreitol (both from New England Biolabs, Ipswich, MA, USA), subjected to gel electrophoresis using BioRad mini-PROTEAN TGX any-kD precast gels and blotted to 0.45 μm PVDF membranes. Membranes were blocked with nonfat dry milk (Carl Roth GmbH, Karlsruhe, Germany) or BSA (Sigma-Aldrich) and incubated with primary antibodies at 4 °C overnight. HRP-linked secondary antibodies and Amersham ECL Prime Detection Reagent (GE Healthcare Dharmacon) were used for detection of proteins on a BioRad ChemiDoc XRS imaging system. RotiFree stripping buffer (Carl Roth GmbH) was used for membrane stripping.

The procedure for immunoblotting was described in detail earlier [7 (link),18 (link)]. Briefly, cells were lysed with 1 × PhosphoSafe lysis buffer (Merck Millipore). Equal amounts of proteins were fractionated by SDS-PAGE on mini-PROTEAN TGX any-kD precast gels (BioRad) and blotted to PVDF membranes. After blockage with nonfat dry milk or bovine serum albumin, membranes were incubated with primary antibodies overnight at 4 °C. After incubation with HRP-linked secondary antibodies, chemiluminescent detection of proteins was performed on a ChemiDoc XRS imaging system (BioRad) using Amersham ECL Prime Detection Reagent (GE Healthcare, Hong Kong, China). Quantification of band intensities for the proteins of interest was carried out by densitometric readings and normalization to those for GAPDH or HSP90 in the same sample.