Trans blot turbo pvdf transfer pack

The colon was collected from rats and flushed of fecal content with ice-cold phosphate-buffered saline (PBS), as described previously [21 , 22 ]. Tissues were minced and homogenized using a Potter–Elvehjem Grinder homogenizer on ice in 20% (w/v) TNE lysis buffer (50 mM Tris–HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 1% NP-40, 1% SDS, 0.1% DOC) with various protease and phosphatase inhibitors. Samples were then sonicated and boiled for 5 min at 95 °C. Protein concentrations were measured with the Bradford assay. Total lysates were run on 4–20% Criterion™ TGX™ Precast Midi Protein Gels (Bio-Rad, Hercules, CA, USA) and then transferred to PVDF membranes (Trans-Blot TurboTM PVDF Transfer packs, Biorad). Then, membranes were blocked with 3% bovine serum albumin (BSA) diluted in Tris-buffered saline (TBS, 20 mM Tris–HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against caspase-1 (ab1872, Abcam) and occludin (ab167161, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by the IBright Analysis software.

Cells were lysed as previously described [23 , 24 ]. Proteins were quantified with the Bradford assay. Proteins were separated onto a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN^® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot^® TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris–HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (ab8227, Abcam), nucleotide-binding oligomerization domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) (ab214185, Abcam), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (D2W8U, Cell Signaling), caspase-1 (ab1872, Abcam), caspase-4 (#4450, Cell Signaling), caspase-5 (#46680, Cell Signaling), and caspase-8 (ab227430, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by the IBright Analysis software.

The colon was collected from rats and flushed of fecal content with ice-cold phosphate-buffered saline (PBS), as described previously [60 (link)]. Tissues were minced and homogenized using a Potter-Elvehjem Grinder homogenizer on ice in 20% (w/v) TNE lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 0.1 mM EDTA, 1% NP-40, 1% SDS, 0.1% DOC) with protease and phosphatase inhibitors. Samples were then sonicated and boiled for 5 min at 95 °C. Proteins were quantified with the Bradford assay. Total lysates were run on a 4–20% Criterion™ TGX™ Precast Midi Protein Gel (Bio-Rad, Hercules, CA, USA) and then transferred to PVDF membranes (Trans-Blot TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against claudin-1 (sc-166338, Santa Cruz, Dallas, TX, USA), occludin (ab167161, Abcam, Cambridge, MA, USA), and ZO-1 (ab96587, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721) phospho-AMPKα (BK2535T, Cell Signaling), AMPKα (BK5831T, Cell Signaling). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad). Densitometry was performed by IBright Analysis software (version, 1.2.2, Waltham, MA, USA).

The colonic tissues were weighed and homogenised in lysis buffer, using a polytron homogeniser, as described by Richter et al. [60 (link)]. Proteins were quantified with the Bradford assay. Proteins (30 μg) were separated onto a pre-cast 4-20% polyacrylamide gel (Mini-PROTEAN^® TGX gel, Biorad) and transferred to PVDF membranes (Trans-Blot^® TurboTM PVDF Transfer packs, Biorad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (monoclonal, diluted 1:5000, A5316, Sigma Aldrich), nucleotide-binding oligomerisation domain leucine rich repeat and pyrin domain containing protein 3 (NLRP3) (polyclonal, diluted 1:1000, ab214185, Abcam), apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (monoclonal, diluted 1:1000, D2W8U, Cell Signaling) and caspase-1 (polyclonal, diluted 1:1000, ab1872, Abcam) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040, Abcam, and anti-rabbit ab6721, Abcam). Protein bands were detected with ECL reagents (Clarity™ Western ECL Blotting Substrate, Biorad). Densitometry was performed by ImageJ software.

Cells were lysed as previously described (Fazzini et al., 2014 (link); Smoktunowicz et al., 2016 (link)). Proteins were quantified with the Bradford assay. Proteins were separated onto a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN® TGX gel, Biorad, Hercules, CA, United States) and transferred to PVDF membranes (Trans-Blot® Turbo^TM PVDF Transfer packs, Biorad, Hercules, CA, United States). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (ab8227, Abcam, Cambridge, United Kingdom), nuclear factor-κB p65 (NF-κB p65, sc-8008, Santa Cruz, Dallas, United States) S100-β (ab52642, Abcam, Cambridge, United Kingdom) and TLR-4 (ab22048, Abcam, Cambridge, United Kingdom) were used. Secondary antibodies were obtained from Abcam (anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity Western ECL Blotting Substrate, Biorad, Hercules, CA, United States). Densitometry was performed by IBright Analysis software.

Cells were lysed as previously described^{62 (link),63 (link)}. Proteins were quantified with the Bradford assay. Proteins were separated onto a pre-cast 4–20% polyacrylamide gel (Mini-PROTEAN^® TGX gel, Bio-rad) and transferred to PVDF membranes (Trans-Blot^® TurboTM PVDF Transfer packs, Bio-rad). Membranes were blocked with 3% BSA diluted in Tris-buffered saline (TBS, 20 mM Tris-HCl, PH 7.5, 150 mM NaCl) with 0.1% Tween 20. Primary antibodies against β-actin (1:5000; ab8227, Abcam), ZO-1 (1:300; ab96587, Abcam), occludin (1:5000; ab31721, Abcam) and TLR-2 (1:500; Santa Cruz, Dallas, TX, USA) were used. Secondary antibodies were obtained from Abcam (1:5000; anti-mouse ab97040 and anti-rabbit ab6721). Protein bands were detected with ECL reagents (Clarity^TM Western ECL Blotting Substrate, Bio-rad). Densitometry was performed by ImageJ software. All blots derive from the same experiment and they were processed in parallel.