Goat anti rabbit hrp

Total protein was extracted from four LA-derived primary cell lines. Protein was separated by SDS-PAGE and transferred to a PVDF membrane as previously described [43 (link)]. The iBind Flex (cat# SLF2000, ThermoFisher Scientific, Waltham, MA, USA) was used for antibody binding with the primary antibodies for OCT4 (1:1000; cat# ab109183, Abcam, Cambridge, UK), NANOG (1:1000; cat# ab109250, Abcam, Cambridge, UK), SOX2 (1:500; cat# 48-1400, ThermoFisher Scientific, Waltham, MA, USA), KLF4 (1:000; cat# NBP2-24749, Novus Biologicus, Centennial, CO, USA), c-MYC (1:1000; cat# ab32072, Abcam, Cambridge, UK) and α-tubulin (1:2000; cat# 62204, ThermoFisher Scientific, Waltham, MA, USA). Secondary antibodies used included goat anti-rabbit HRP (1:1000; cat# ab6721, ThermoFisher Scientific, Waltham, MA, USA) and goat anti-mouse alexa488 (1:1000; cat# A21202, ThermoFisher Scientific, Waltham, MA, USA). Clarity Western ECL (cat# 1705061, Bio-Rad, Los Altos, CA, USA) was used for visualizing HRP detected protein bands and the ChemiDoc MP Imaging System (Bio-Rad Laboratories, Los Altos, CA, USA) and Image Lab 6.0 software (Bio-Rad Laboratories, Los Altos, CA, USA) were used for band detection and analysis. NTERA-2 cells were used as a positive control, and α-tubulin was used as a loading control.

Proteins were resolved through SDS-PAGE using NuPAGE MOPS SDS running buffer (Thermo Fisher Scientific) and 4-15% precast Mini-PROTEAN TGX Gels (Bio-Rad) before transferring to a PVDF membrane (Bio-Rad). Immunodetection was achieved with 1:2,500 anti-myc (Cell Signaling Technology), 1:20,000 anti-FLAG (Sigma-Aldrich), and 1:20,000 anti-actin-HRP (Sigma-Aldrich). Primary antibodies were detected with 1:20,000 goat anti-mouse HRP (Jackson ImmunoResearch) or 1:20,000 goat anti-rabbit HRP (Thermo Fisher Scientific). Proteins were visualized on a ChemiDoc (Bio-Rad) using ProSignal Pico ECL Reagent (Genesee Scientific). ImageJ was used to quantify western blots as previously described (Davarinejad, 2015 ). Briefly, protein band intensities for each blot were measured by taking the net grey mean value of each band. The net grey mean value is defined as the inverted pixel density (255 – grey mean value) of a band with the inverted pixel density of the background (defined as an equivalent area of the blot above or below the band) subtracted.

Protein expression was evaluated by Western blot analyses. Samples were homogenized in RIPA buffer (150 mM NaCl, 10 mM Tris, pH 7.2, 0.1% SDS, 1% Triton-X100, 5 mM EDTA, 100 μM Na₃VO₄, 10 mM NaF), and Protease inhibitor 1X (Thermo Fisher Scientific), loaded onto a 4–10% acrylamide gradient gel, separated by electrophoresis, and transferred to a nitrocellulose membrane (Millipore). Primary antibodies against the following proteins were used: Ca_vα1.2 (Abcam), GAPDH (14C10) (Cell Signaling Technology). Goat anti-mouse-HRP and Goat anti-rabbit-HRP (Thermo Fisher Scientific) were used as secondary antibodies. ECL (Millipore) was used for protein detection using a Chemidoc MP Imaging System (Biorad). Image J software (National Institutes of Health) was used for densitometry analysis.

For immunoblots, the descending colon samples (N = 6 for both genotypes) were homogenized in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 25 mM Tris (pH 7.4), 1 mM sodium metabisulphite , 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) using plastic pestles. The lysates were centrifuged at 12,000×g at 4 °C for 30 min, the supernatants were collected and used to quantify protein concentration as described by Bradford^{91 (link)}. Total protein extracts were boiled in SDS-PAGE sample buffer, and about 40 μg of total protein was loaded per lane of the 15% acrylamide gel. Rabbit polyclonal anti-Claudin-3 (ab15102, Abcam, Cambridge, UK), rabbit polyclonal anti-Claudin-7 (34-9100, ThermoFisher Scientific, Waltham, MA, USA), rabbit polyclonal anti-β-actin (#PA5-16914, ThermoFisher Scientific, Waltham, MA, USA), and mouse monoclonal anti-GAPDH (MA5-15738, ThermoFisher Scientific, Waltham, MA, USA) antibodies were used at 1:1000. Goat anti-rabbit HRP and goat anti-mouse HRP (#A-11036 and #G-21040 respectively, both ThermoFisher Scientific, Waltham, MA, USA, 1:3500) served as secondary antibodies. Images were captured using an Amersham Imager 600 System (GE Healthcare) and Novex ECL Chemiluminescent Substrate Reagent Kit (ThermoFisher Scientific, Waltham, MA, USA). Immunoblot quantification was performed using ImageJ software.

Total protein concentrations from COS, OPC, and GT1–7 cell lysates were determined via BCA assay kit (Pierce Biotechnologies, Rockford, IL). Plates were read using a SkanIt plate-reader (Thermo Fisher Scientific, Waltham, MA) at absorbance of 562 nm. Twenty-five micrograms of protein were separated on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Primary antibodies for GPR54/Kiss1R (Alamone, Jerusalem, Israel) were used to probe blots. Bound primary antibody was probed by goat anti-rabbit HRP and detected with Supersignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA). The blots were scanned on an ImageQuant LAS4000 (GE Healthcare Life Sciences, Marlborough, MS). Anti-alpha-tubulin followed by goat anti-rabbit HRP (Santa Cruz Biotechnology, Dallas, TX) were used to assess protein loading and normalize band intensities.