Anti β actin peroxidase

The following antibodies have been used throughout this study: anti-β-Catenin (Cell Signaling, #8480), anti HA-Peroxidase (Sigma Aldrich, #34071100), anti rabbit IgG-Peroxidase (Abcam, ab6721; secondary antibody, Western Blot), anti mouse IgG-Peroxidase (Abcam, ab97023; secondary antibody, Western Blot), anti GFP (Abcam, ab290), anti UBF (Santa Cruz, sc-13125), anti mCherry (Abcam, ab125096), anti β-Actin-Peroxidase (Sigma Aldrich, A3854), anti RNA Polymerase II(Diagenode, AC-055-100), anti mouse IgG-Alexa Fluor®586 (Abcam, ab175473; secondary antibody, IHC), anti p53 (Santa Cruz, sc-47698), Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) (Abcam, ab5095), Goat Anti-Rabbit IgG H&L (FITC) (Abcam, ab6717) respectively.

Immunoblotting was performed using the Novex NuPAGE SDS-PAGE Gel and blotting system (Invitrogen). Antibodies were anti-total HSL, anti-p565 HSL and anti-660 HSL (sampler kit #8334; Cell Signalling Technology, NEB, Herts, UK), anti-FABP4 (HPA002188; Sigma-Aldrich, St Louis, MO, USA) and anti-β-actin-peroxidase (Sigma-Aldrich).

Equal number of the peritoneal macrophages were seeded in 24-well plate and infected with different Mtb strains at an MOI of 1:10. At different time points, cells were washed and lysed in RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml each of aprotinin, leupeptin, pepstatin, 1 mM Na₃VO₄, 1 mM NaF] on ice for 30 min and total protein was collected. An equal amount of protein from each cell lysate was electrophoresed on SDS-PAG and transferred onto PVDF membrane (Millipore, USA) by semi-dry Western blotting method (Bio-Rad, USA). Non-specific binding was blocked with 5% non-fat dry milk powder in TBST [20 mM Tris-HCl (pH 7.4), 137 mM NaCl, and 0.1% Tween 20] and incubated overnight at 4°C with primary antibody diluted in TBST with 5% BSA. The blots were washed and incubated with anti-rabbit or anti-mouse secondary-HRP conjugated antibodies for 2 h. The blots were developed using enhanced chemiluminescence detection system (Perkin Elmer, USA) as per manufacturer's instructions. β-ACTIN was used as a loading control. Phospho-mTOR (Ser2448) and HRP-conjugated anti-rabbit IgG antibodies were from Cell Signaling Technology and Jackson Immuno Research, USA, respectively. Anti-β-ACTIN-Peroxidase and anti-LC3B antibodies were from Sigma-Aldrich, USA.

Protein expression of NLRP3 and caspase-1 was determined using protein analysis of thoracic aortas of in vivo and in vitro all groups. Samples were homogenized in lysis buffer and proteins were collected. Proteins (30 μg) were separated by electrophoresis on 10 or 12% polyacrylamide gels, transferred to 0.22 μm nitrocellulose membranes and blocked using 5% bovine serum albumin (BSA) in Tris buffered saline (TBS) and 0.1% Tween 20 for 1 h. Primary antibodies were incubated overnight at 4°C as follows: anti-NLRP3 (1:500 dilution; R&D Systems), anti-caspase-1 (1:1,000 dilution; Novus Biologicals), anti-β-actin-peroxidase (1:5,000 dilution; Sigma-Aldrich).

Total lysates for the immunoblotting analyses were obtained as follows: the primary hippocampal neurons were washed in phosphate buffered saline (PBS) and collected by gentle scraping in ice-cold RIPA buffer containing (in mM): 50 Tris pH 7.4, 100 NaCl, 1 EGTA, 1 PMSF, 1 Na₃VO₄, 1 NaF, 0.5% NP-40, and 0.2% SDS, and supplemented with protease inhibitor cocktail II (Roche Diagnostic, Monza, Italy). The nitrocellulose membranes were incubated with the following antibodies: rabbit K_V2.1 antibody (1:1000, Alomone Labs, Jerusalem, Israel), mouse α-tubulin antibody (1:5000, Sigma-Aldrich, Milan, Italy), and anti-β-actin peroxidase (1:10,000, Sigma-Aldrich, Milan, Italy). The immunoreactive bands were detected with the chemiluminescence system (GE Healthcare, Milan, Italy). The films were developed with a standard photographic procedure, and the quantitative analysis of the bands detected was carried out by densitometric scanning.

Whole-cell lysates were homogenized in modified RIPA buffer^{14 (link)} and assayed by immunoblot as previously described^{14 (link)}. The primary antibodies against FKBP51 (rabbit polyclonal; Novus Biological), FKBP51s^{13 (link)} and CD274/PD-L1 (rabbit polyclonal; Novus Biological) were used diluted 1:2500. CD133 (rabbit polyclonal; Abcam; Cambridge, UK) was used diluted 1:1000. A further antibody Pdcd-1L1 (rabbit polyclonal, Santa Cruz Biotechnology; Santa Cruz, CA, USA) was used for PD-L1 detection at the 1:1000 dilution. Antibody against M2-Flag, caspase-7 and γ-Tubulin (mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA) were used diluted 1:5000. Anti β-Actin-Peroxidase (mouse monoclonal; Sigma-Adrich) was used diluted 1:10000. Anti-phospho-Akt (Ser473), Akt1/2/3, phosphor-S6 kinase, G3PDH and Sox-2 (rabbit monoclonal; Cell Signaling, Danvers, MA, USA) were used diluted 1:1000. Anti-p70S6 kinase (rabbit polyclonal; Santa Cruz) was used diluted 1:500.