Amersham ecl plus western blotting detection system

Samples were separated by SDS-PAGE on a 7.5% (w/v) gel and transferred to a Protran nitrocellulose membrane (Whatman, Dassel, Germany). The membrane was blocked in 5% bovine serum albumin (BSA) in TBS-Tween (0.05% (w/v) Tween-20 in 10 mM Tris, 100 mM NaCl, pH 7.5), and then incubated with the primary antibody (anti-HA-HRP, 1∶750) in 1% BSA/TBS-Tween. Membranes were developed using the Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). Bands were quantified with ImageJ.

For western blot analysis, Caco-2 monolayers were grown on 6-well cell-culture plates (corning) until 100% confluency. Monolayers were then incubated with 1 × 10⁸ parasites/mL for 6 and 12 h, respectively, washed with PBS twice, and then scraped and collected. Monolayers were then incubated with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Pierce). Lysed samples were centrifuged at 21,000 rpm at 4°C for 30 minutes. Protein concentration of the supernatant was determined with the D_C Protein Assay (Bio-Rad Laboratories). SDS-PAGE gels (12% and 15% Tris-HCL Ready-Gels; Bio-Rad Laboratories) were used to separate total proteins. Proteins were then transferred using a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore). 5% of nonfat dry milk in % TBS-T was then used to block the membranes. After blocking, membranes were incubated with primary antibodies against, caspase 3, caspase 8, caspase 9, or ZO-1 (1 : 1000; Sigma) overnight at 4°C. After incubation with primary antibodies, membranes were washed and incubated with HRP-tagged secondary antibodies. Bands were detected using Amersham ECL Plus western blotting detection system (GE Healthcare). Autoradiographic films (Kodak) were then exposed to the membranes and developed on X-ray film processor SRX-101A (Konica Minolta).

Total protein was extracted from cell pellets. The protein concentrations were determined using the BCA protein assay (Thermo Fisher Scientific, Rockford, USA). Then, thirty micrograms of each sample was separated by 10% SDS–polyacrylamide gel electrophoresis and transferred to a PVDF membrane (0.22 micrometer, Bio-Rad). The membrane was then blocked with 5% skim milk at room temperature. Rabbit polyclonal anti-α-SMA, Collagen I, p65, P-p65 and UBC9 (Abcam, USA)) were diluted 1:1000. Bax and Bcl-2 (Proteintech, Wuhan, China) were diluted 1:200. Rabbit monoclonal antibodies against Caspase 3 and cleaved-Caspase3 (cell Signaling, USA) were diluted 1:1000, and a mouse monoclonal antibody directed against β-actin (Proteintech, Wuhan, China) was used at 1:1000. The membrane was incubated with these antibodies overnight at 4°C. The membranes were washed three times with TBS/Tween 20 (0.075%) containing 3% Marvel for 15 min each before incubation with HRP-conjugated secondary antibodies (1:10,000) at 37°C for 1 h. The PVDF membrane was washed in TBST three times (10 min each time). The proteins were visualized using the Amersham^™ ECL Plus Western Blotting Detection System (GE Healthcare, UK).

The level of histone acetylation/lactylation was determined by immunoblot analysis, a semi-quantitative technique widely used in molecular biology and biochemistry research. HeLa cells were seeded in a dish, and after 48 h they were treated for 24 h with L. crispatus or L. reuteri supernatant diluted 1:100 in culture medium. Cells were harvested and washed with 10 mM sodium butyrate in PBS, and nuclei were isolated in according to Amellem et al. [21 (link)]. The nuclear histones were extracted as previously described [22 (link)]. Histones were detected resolving samples on a 10% gel in MES buffer at 200 V for 40 min. Western Blotting was performed in transfer buffer at 100 V for 1 h. The nitrocellulose membrane was incubated with primary antibody specific for anti-acetylated lysine (Millipore, Billerica, MA, USA) for 1 h. After washes with PBS-TWEEN 20 0.1%, the membrane was incubated as before with secondary horseradish-peroxidase-conjugated antibody (GE Healthcare, Milan, Italy). After washes with PBS-TWEEN 20 0.1%, antibody binding was detected using an Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Milan, Italy). Densitometry analysis was performed with a Fluor-S Max MultiImager (Bio-Rad, Hercules, CA, USA), and relative quantification of histone acetylation signals was carried out by using densitometry and normalized on H1 signal as a control.

MDA-MB-231 cells treated with TNFα at different time points were harvested and incubated for 30 min with lysis buffer (10X Lysis Buffer, Cell Signaling, Danvers, MA, USA). The protein lysates were prepared and resolved by 12% SDS-PAGE as described earlier [38 (link)]. Cellular proteins were transferred to an Immuno-Blot Polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories, Hercules, CA, USA) by electroblotting. The membranes were then blocked with 5% non-fat milk in PBS for 1 h, followed by incubation with primary antibodies against p44/42 MAPK (ERK1/2; cat# 9101) and 44/42 MAPK (ERK1/2; cat# 9102), p-p38 (cat# 9211) and p38 (cat# 9212), p-NF-κB (cat# 3033) and NF-κB (cat# 3034), and β-actin (cat# 4967) in 1:1000 dilution at 4 °C overnight. All primary antibodies were purchased from Cell Signaling (Cell Signaling Technology Inc., Danvers, MA, USA). The blots were then washed three times with TBS-T and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA). Immunoreactive bands were developed using an Amersham ECL Plus Western Blotting Detection System (GE Healthcare, Chicago, IL, USA) and visualized by Molecular Imager® VersaDoc^TM MP Imaging Systems (Bio-Rad Laboratories, Hercules, CA, USA).

Following different treatments, monocytic cells were harvested and incubated for 30 min with lysis buffer (10× Lysis Buffer, Cell Signaling Technology Inc., Danvers, MA, USA). The protein lysates were prepared and resolved using 12% SDS-PAGE as described earlier [44 (link),52 (link)]. Cellular proteins were transferred to Immuno-Blot PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) by electro blotting. The membranes were then blocked with 5% non-fat milk in PBS for 1 h followed by incubation with primary antibodies against p-JNK and JNK, p-P38 and P38, p-ERK and ERK, and p-NF-κB and NF-κB at a 1:1000 dilution at 4 °C overnight. All the primary antibodies were purchased from Cell Signaling (Cell Signaling Technology Inc., Danvers, MA, USA). Anti-MCP1 antibody was bought from abcam (abcam, Cambridge, MA, USA), and B-actin was purchased from Cell Signaling Technology, Inc. The blots were then washed three times with TBS-T and incubated for 2 h with HRP-conjugated secondary antibody (Promega, Madison, WI, USA). Immunoreactive bands were developed using an Amersham ECL Plus Western Blotting Detection System (GE Health Care, Buckinghamshire, UK) and visualized with a Molecular Imager^® (VersaDocTM MP Imaging Systems, Bio-Rad Laboratories, Hercules, CA, USA) [53 (link)].