Precision plus protein dual xtra prestained protein standard

The protein profiles of LPI and its fractions were analyzed using SDS-PAGE in Criterion automated equipment (Bio-Rad Laboratories) using 12% Criterion™ TGX™ Precast Midi Protein Gels (Bio-Rad). Samples (1 mg of protein/mL) were dissolved in a sample buffer containing 0.05 M Tris-HCl (pH 6.8), 1.6% (p:v) SDS, 8% (v:v) glycerol, 0.02% (p:v) 2-bromophenol, and 2% (v:v) β-mercaptoethanol, heated for 5 min at 100 °C, and cooled to room temperature. A Precision Plus Protein™ Dual Xtra Prestained Protein Standard (Bio-Rad Laboratories) was used as a molecular weight marker. Next, 40 µL of the sample was loaded onto the gel and run with voltages of 100 V (5 min) and 150 V (50 min). The commercial buffer XT MES Running Buffer 20X (Bio-Rad Laboratories) was used for the separation. The gel was stained with InstantBlue^® Commassie Protein Stain (Abcam, Cambridge, UK). A gel image was taken using the Versadoc Imaging System gel reader and analyzed using software image lab 6.1 (Bio-Rad Laboratories).

The molecular mass of the purified bacteriocin was estimated by Tricine SDS PAGE 16.5% gel and Zymogram [17 (link),18 (link)]. The aliquot of purified bacteriocin and crude extract (CRE) were loaded in duplicate wells along with the Precision plus protein™ dual Xtra prestained protein standard (Bio-Rad Laboratories Inc., Hercules, CA, USA). The samples were electrophoresed in Tris-Tricine buffer solution at 10 mA for 4 h (Cleaver Scientific Ltd., Rugby, UK). The gel was divided so that each half contained both samples. One half-gel was stained with Coomassie blue, followed by a destaining step. Another half was used to detect the in-gel antimicrobial band by a Zymogram assay. It was fixed in a fixing solution (40% ethanol and 10% acetic acid) for 30 min, washed in water for 2 h, and placed onto a 7-mm layer of Mueller–Hinton agar (0.8% agar, 3% NaCl) inoculated with V. parahaemolyticus cells (~10⁶ CFU/mL). Another 7 mL of melting Mueller–Hinton ½ strength agar (0.8% agar, 3% NaCl) inoculated with V. parahaemolyticus cells was poured over the gel. After solidification, the plate was incubated for 8–12 h at 30 °C. An inhibition zone appeared in the gel if the samples contained bacteriocin.

Whole lysates of control and IPF fibroblasts were harvested in RIPA buffer (Sigma-Aldrich, R0278) supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, 78430). SDS-PAGE electrophoresis was used to separate proteins using the Mini-PROTEAN® TGX™ Precast Gels (Bio-Rad, 4561085) with Precision Plus Protein™ Dual Xtra Prestained Protein Standards (Bio-Rad, 1610377). Samples were transferred to PVDF membranes using the Trans-Blot® Turbo™ Transfer System (Bio-Rad) and the membranes were blocked for 1 h at room temperature (RT) in blocking buffer (3% BSA in PBS) before they were incubated overnight at 4 °C in a 3% BSA-TTBS (Tris-buffered saline with Tween) blocking buffer with the following antibodies: β-actin (BioLegend, 643802), H3K4me1 (Diagenode, C15410194), H3K4me3 (Diagenode, C15410003) and H3K27ac (Diagenode, C15410196). The membranes were washed 3 times in TTBS for 5 mins and incubated with IRDye® 800CW Goat anti-Mouse (LI-COR, 926-32210) and IRDye® 680RD Donkey anti-Rabbit (LI-COR, 926-68073) for 1 h at RT before they were washed and imaged on the LI-COR Odyssey CLx imaging system.