The HPAECs, EGM, FBS growth supplement, HEPES, 0.25% Trypsin-EDTA, and trypsin-neutralizing solution were used as supplied by Clonetics (San Diego, CA, USA). The HK and PK were purchased from Enzyme Research Laboratory (South Bend, IN, USA). S2302 was purchased from DiaPharma (Franklin, OH, USA). The primary antibody, Goat Anti-Human PRCP, and the secondary Anti-Goat IgG: Whole Molecule, Peroxidase Conjugate were purchased from Bioscience (Long Beach, CA, USA). Mouse Anti-Human β-Actin was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The “Nitrate/Nitrite Fluorometric Assay Kit” was purchased from Cayman Chemicals (Ann Arbor, MI, USA). The Cellular Senescence Assay Kit was purchased from Chemicon International (Temecula, CA, USA). TRIzol, Super Signal West Femto Maximum Sensitivity Substrate, RiPA Buffer, Super Signal West Femto Maximum Sensitivity Substrate were supplied by ThermoFisher Scientific (Rockford, IL, USA). The Precision Plus Protein standard (Dual Color) was purchased from Bio-Rad (Rockford, IL, USA). Ethidium bromide was from Sigma-Aldrich (St. Louis, MO, USA).
Supersignal west femto maximum sensitivity substrate
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, United Kingdom, Germany, China, France, Australia, Switzerland, Canada, Denmark, Israel
SuperSignal West Femto Maximum Sensitivity Substrate is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It provides high sensitivity detection with a wide linear dynamic range.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (142 mentions)
Pvdf membrane, by Merck Group (68 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (67 mentions)
Trans blot turbo transfer system, by Bio-Rad (60 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (51 mentions)
Ripa buffer, by Thermo Fisher Scientific (49 mentions)
Chemidoc mp imaging system, by Bio-Rad (46 mentions)
Nitrocellulose membrane, by Bio-Rad (42 mentions)
Image lab software, by Bio-Rad (40 mentions)
Chemidoc imaging system, by Bio-Rad (38 mentions)
Protease inhibitor cocktail, by Merck Group (34 mentions)
Chemidoc xrs system, by Bio-Rad (33 mentions)
β actin, by Merck Group (33 mentions)
Pvdf membrane, by Bio-Rad (33 mentions)
Chemidoc touch imaging system, by Bio-Rad (30 mentions)
Imagequant las 4000, by GE Healthcare (28 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (27 mentions)
Gapdh, by Cell Signaling Technology (27 mentions)
Protease inhibitor cocktail, by Roche (27 mentions)
β actin, by Cell Signaling Technology (25 mentions)
1 483 protocols using supersignal west femto maximum sensitivity substrate
1
Protein Expression and Senescence Assay
2
Western Blot Analysis of Signaling Pathways
3
Immunoblotting and qPCR for Protein and Gene Analysis
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Immunoblotting and qPCR were carried out as previously reported (Yang et al., 2021 (link)). Briefly, for immunoblotting, cells were lysed with LDS sample buffer (Thermo Fisher Scientific) and subjected to SDS-PAGE followed by transferring to a PVDF membrane. The blots were then probed with indicated antibodies and bands were visualized with SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific). For S protein incorporation test, pseudoviruses were first ultracentrifuged as described above and then subjected to SDS-PAGE. To quantify phosphatidylinositol 4-kinase β (PI4KB) mRNA, total RNAs from control or shRNA transduced cells were extracted with GeneJET RNA Purification Kit and then reversed transcribed with random primer and RevertAid Reverse Transcription kit (Thermo Fisher Scientific). Quantification was performed by real-time quantitative PCR with SYBR Mix (Genstar qPCR Master Mix) using specific primers listed in Supplementary Table S2 .
4
Protein Quantification and Immunoblotting Analysis
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Determination of the protein concentration was performed by Coomassie Protein Assay (Thermo Scientific). All samples were diluted in Laemmli Loading Buffer (WVR Life Science). The immunoreactivity signals were detected by Super SignalTMWest Femto Maximum Sensitivity Substrate (Thermo Scientific) using: two different rabbit polyclonal anti-STX-1a, rabbit polyclonal anti-ERp57, rabbit polyclonal anti-CD9, rabbit polyclonal anti-SNAP-25; rabbit polyclonal anti-Lamin A/C, rabbit monoclonal anti-SV2A, rabbit monoclonal anti-GFAP, rabbit monoclonal anti-PSD95, mouse monoclonal anti-β-actin (ACTB), rabbit polyclonal anti-APO A1, rabbit polyclonal anti-NSE, mouse monoclonal anti-GM130 and mouse monoclonal anti-CD107a. The informations of all antibodies used have been reported in Supplementary table 1 online. Images were acquired using Azure C300 Gel Imaging System (Bio-System) and WB densitometric analyses were performed using Image J software (Meida Cybernetics).
5
Western Blot Analysis of Proteins
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Protein samples were analyzed via Western blotting as described previously [17 (link)]. Proteins (whole cell extracts, 30 μg/lane) were separated via electrophoresis in NuPAGE 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA), blotted onto PVDF transfer membranes, and analyzed using epitope-specific primary and secondary antibodies. Bound antibodies were visualized using SuperSignalTM WestFemto Maximum sensitivity substrate (Thermo Fisher Scientific, San Jose, CA, USA) and an LAS 4000 imaging system (Fujifilm, Japan). The primary and secondary antibodies were used as indicated in Table S1 .
6
Western Blot Analysis of Drosophila Proteins
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Flies were snap-frozen in liquid nitrogen, and their heads were collected and homogenized in RIPA buffer (10 µl/head) using a Speedmill P12 (Analytik Jena AG). The total protein concentration was measured with a DCTM Protein Assay Kit (Bio-Rad Laboratories, USA). Twenty micrograms of protein was separated via SDS-PAGE before being blotted onto nitrocellulose membranes. The membranes were blocked with 5% skim milk in TBS-T, after which they were incubated with the primary antibodies overnight at 4 °C. Incubation with the corresponding HRP-conjugated secondary antibody was carried out for 2 h at room temperature and followed by signal detection via chemiluminescence (SuperSignalTM West Femto Maximum Sensitivity Substrate, Thermo Scientific, Rockford, USA). The following antibodies were used: mouse anti-GFP (1:1000 #11814460001, Roche Diagnostics) and mouse anti-Drosophila CSP-2 (1:500, #6D6, Developmental Studies Hybridoma Bank, IA USA).
7
Cochlear Protein Extraction and Analysis
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At least 10 cochleae were dissected and homogenized in 100 μl ice-cold RIPA lysis buffer (Beyotime, #P0013B) using a tissue homogenizer (Shanghai Jingxin Industrial Development Co., Ltd., #JXFSTPRP-48). After centrifuging at 12,000 × g for 15 min at 4°C, the supernatant was boiled with 5 × SDS loading buffer, separated by 10% SDS-PAGE, and transferred to an Immobilon PVDF membrane (Millipore, #ISEQ00010). The membrane was first blocked for 1 h at room temperature with 5% non-fat dried milk in 0.1% PBS-Tween 20, incubated with the anti-Strip1 (Novusbio, # NBP2-45715) and anti-β-actin (Abcam, #ab119716) primary antibodies at 4°C overnight, and then incubated by HRP-conjugated secondary antibodies (Abmart, goat anti-mouse HRP, #M21001 and goat anti-rabbit HRP, #M21002) for 1 h at room temperature. The SupersignalTM West Femto Maximum Sensitivity Substrate (Thermo, #34,094) was used to detect the signals on a FluorChem M system (ProteinSimple, #FM0477).
8
Western Blot Analysis of Celf1 and p27
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Lenses from control and Celf1cKO/lacZKI animals were dissected and homogenized in ice-cold lysis buffer (50mM Tris-HCl at pH 8, 150mM NaCl, 1% nonidet P40, 0.1%SDS, 0.5% sodium deoxycholate, along with protease inhibitors (Thermo Fisher Scientific, Waltham, MA). For cell line lysate preparation, lysis buffer (1 mL) was directly added to the cell culture plate and incubated at 4°C for 30 min. Cell debris was removed by centrifuging lysates at 14,000 RPM for 30 min at 4°C. Protein concentration was determined using Pierce BCA protein kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Total protein (25–50 μg) was resolved on TGX stain free polyacrylamide gels (Bio-Rad, Hercules, CA Hercules, CA) and transferred onto PVDF membrane (Thermo Fisher Scientific, Waltham, MA). Blots were blocked with 5% non-fat dry milk for 1 hour at room temperature and incubated with primary antibody (p27Kip1 BD Bioscience, San Jose, CA) 610241 and Celf1 ab-9547 at 1:500 and 1:1000 dilutions, respectively) over night at 4°C. Blots were incubated with secondary antibodies conjugated to horseradish peroxidase (Cell Signaling Technology, Danvers, MA) for one hour at room temperature, and the signals were detected with SuperSignalTM West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, Waltham, MA).
9
Western Blot Analysis of Tight Junction Proteins
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Total protein lysates were collected either from colon tissue or colon epithelial cells as described above using radioimmunoprecipitation assay (RIPA) buffer (Millipore Sigma, St. Louis, Missouri, USA) and quantified using BCA protein quantification kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) per instructional manual. Total protein (20–50 μg) of was resolved on Mini-PROTEAN TGX 4–20% gels (Bio-Rad, Hercules, California, USA) and transferred to polyvinylidene difluoride membrane (0.22 μm pore; Novex, Carlsbad, California, USA). After blocking with 3% (w/v) bovine serum albumin (BSA) (containing 1X TBS) for 1 h, the membrane was then incubated with HRP-conjugated anti-claudin-4, anti-occludin, anti-IL-10RA and anti-β-actin antibodies (1:500, 1:500, 1:300 and 1:20,000 dilution, respectively) at 4°C overnight. For all proteins, chemiluminescent substrate (SuperSignalTM West Femto Maximum Sensitivity Substrate, Thermo Scientific, Rockford, Illinois, USA) was used to detect the protein bands (ImageQuant LAS 4000). Densitometry analysis of bands was done using ImageJ software. Antibodies for claudin-4, occludin and β-actin were purchased from Santa Cruz Biotechnologies (USA). The antibody for IL-10RA was purchased from Novus Biologicals (USA).
10
Protein Extraction and Western Blot Analysis
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Protein extraction from dissected tissues was performed as previously described [70 (link)]. Extracts were separated by electrophoresis in a 10% SDS-polyacrylamide gel at constant current of 120 volt. Proteins were transferred to polyvinylidinefluoride membranes (Millipore Corporation, Billerica, MA, USA), subsequently blocked 5% dry milk in TBST (Tris Buffered Saline with 0.1% Tween 20) for 1 h at room temperature and then incubated with anti-Dfr S/L, anti-Phm, anti-Dib, or anti-Actin (mAbcam 8224) as primary antibodies, and with ECLTM anti-rat IgG (Amersham), ECLTM anti-mouse IgG (GE Healthcare), and ECLTM anti-rabbit IgG (GE Healthcare) as 2nd antibodies. The blot was developed using either SuperSignalTM West Femto maximum sensitivity substrate or SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific) according to the manufacturers’ instructions. Digital images were acquired with ChemiDoc™ Imaging Systems (Bio-Rad). Protein levels were quantified with Image Lab™ Software (Bio-Rad) and normalized against Actin or Lamin. Statistics was performed using two-way ANOVA.
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