Twenty guts from adult flies were dissected in ice-cold PBS, and homogenized in 50-μl lysis buffer followed by mixing with 5× loading buffer as described previously [33 (link)]. Ten microliters of total lysates were subjected to Western blotting. The primary antibodies were anti-α-syn (Novus Biologicals, 1:2000 ) and anti-α-actin (Millipore, 1:50,000). The processed membrane was developed with chemiluminescent HRP substrate (Millipore, 1:1000) to detect target proteins.
Chemiluminescent hrp substrate
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, China, Japan, France
Chemiluminescent HRP substrate is a laboratory reagent used to detect and quantify the presence of horseradish peroxidase (HRP) in various assays. It generates a luminescent signal when catalyzed by HRP, allowing for sensitive and accurate measurement of HRP-labeled targets.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (86 mentions)
Bca protein assay kit, by Beyotime (18 mentions)
Ripa lysis buffer, by Beyotime (17 mentions)
Polyvinylidene fluoride membrane, by Merck Group (14 mentions)
Ripa buffer, by Beyotime (14 mentions)
Chemidoc xrs system, by Bio-Rad (13 mentions)
Protease inhibitor cocktail, by Roche (13 mentions)
Gapdh, by Cell Signaling Technology (11 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (11 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (9 mentions)
Image lab software, by Bio-Rad (9 mentions)
Polyvinylidene difluoride membrane, by Merck Group (8 mentions)
Pvdf membrane, by Bio-Rad (8 mentions)
Ripa lysis buffer, by CWBIO (8 mentions)
Gapdh, by Santa Cruz Biotechnology (8 mentions)
Nitrocellulose membrane, by Bio-Rad (7 mentions)
Anti β actin, by Santa Cruz Biotechnology (6 mentions)
Gapdh, by Abcam (6 mentions)
Protease inhibitor, by Merck Group (6 mentions)
Amersham imager, by Cytiva (6 mentions)
389 protocols using chemiluminescent hrp substrate
1
Protein Extraction from Fly Gut Tissues
2
FHR1 Impact on Complement Factor I Activity
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To investigate the effect of FHR1 on the cofactor activity of FH, the generation of C3b cleavage products by complement factor I (CFI) was assessed by SDS-PAGE and Western blotting. Two serially diluted isoforms of FHR1 (125 μg/ml-62.5 μg/ml), alone or in combination with FH (15.64 ng), were mixed with C3b (1.1 μg) and CFI (100 ng) and then incubated at 37°C for 30 min. After SDS-PAGE and Western blotting, the C3b degradation product (α’ 43 kDa) was detected with a C3c polyclonal antibody (Dako), followed by an HRP-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Labs). The membranes were developed with a chemiluminescent HRP substrate (Millipore), and the signals of the C3b degradation product (α’ 43 kDa) were analyzed by ImageJ.
3
Western Blot Protein Analysis Protocol
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All samples were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM sodium orthovanadate, 1× sigma protease inhibitor cocktail) in ice for 30 min. Lysates were then quantified via Pierce® BCA Protein Assay Kit (Thermo scientific, Rockford, IL, USA). In total, 10–50 μg of protein was separated by 10–15% SDS-PAGE and then transferred onto Immobilon®-P membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk/TBS-T for 1 h and incubated with primary antibodies for overnight at 4 °C. After washed three times, the membranes were incubated with HRP-conjugated secondary antibodies for 40 min at room temperature. After 1.5 h washing, protein bands were visualized using Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).
4
Western Blot Protein Analysis Protocol
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Tissue or cell lysates with the same protein content (assayed by the BCA method; Bio‐Rad, CA) were prepared. Proteins were separated by 10% SDS‐PAGE or 12% SDS‐PAGE and transferred to a polyvinylidene difluoride membrane (Millipore). The membranes were blocked for 1.5 hours in 3% milk and then incubated overnight at 4°C with primary antibodies, followed by horseradish peroxidase–conjugated rabbit anti‐goat (1:5000) or goat anti‐rabbit immunoglobulin G (1:5000 or 1:10 000) for 2 hours at room temperature. The bands were scanned and detected by a standard enhanced chemiluminescence method with Chemiluminescent HRP Substrate (Millipore, WBKLS0100). ImageJ Software was used to quantitate the intensity of the bands.
5
Western Blot Analysis of Histone Modifications
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Whole cells or human endometrial tissues were lysed using radioimmunoprecipitation assay buffer (Thermo Fisher Scientific) including a protease inhibitor cocktail (ThermoFisher Scientific). About 20 μg protein extract were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were blocked in 5% skimmed milk at room temperature for 1 h and then incubated with primary antibodies against NSD2 (1:1000; #65127, Cell Signaling Technology), H3K9me3 (1:1000, ab176916, Abcam), H3K27me3 (1:1000, ab192985, Abcam), H3K36me1 (1:1000, ab176920, Abcam), H3K36me2 (1:1000, ab176921, Abcam), H3K36me3 (1:1000, ab176916, Abcam), and β-actin (ACTB; 1:10,000, 66009-1-Ig, Proteintech, Rosemont, IL, USA) at 4°C overnight. Next day, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The membranes were visualized using Chemiluminescent HRP Substrate (Millipore). The immunoreactive protein band density was quantified using Image J software (NIH). Raw data of all western blots are provided in Supplementary Fig. 4.
6
Western Blot Analysis of FAS Protein
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Whole-cell extracts of cultured cells were isolated in with lysis and extraction buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1.5 mM MgCl2 and 1% Triton X-100). Cell debris was removed by centrifugation for 20 min at 12,000g at 4 °C in a microcentrifuge. Lysate was mixed with 5× SDS loading buffer and heated for 5 min at 100 °C. Prepared samples were resolved by 10% SDS–PAGE and transferred to PVDF membranes (Bio-Rad). The membrane was blocked for 1 h at room temperature using blocking buffer (5% nonfat dried milk diluted in TBST). Blots were incubated with anti-FAS monoclonal antibody (3180S, clone C20G5, Cell Signaling Technology) diluted at 1:1,000. Protein expression was normalized to β-actin (sc-47778, clone C4, Santa Cruz Biotechnology) diluted at 1:2,000. The HRP-conjugated anti-mouse IgG antibody (31430, clone 31430, Thermo Fisher Scientific) and the HRP-conjugated anti-rabbit IgG antibody (32460, clone 32460, Thermo Fisher Scientific) were used to detect primary antibodies at 1:10,000 dilution. HRP was detected using chemiluminescent HRP substrate (Millipore). Digital images were acquired with the ChemiDoc XRS+ System and analyzed by Image Lab v.5.1 (Bio-Rad).
7
Quantification of Mitochondrial Proteins
8
Western Blot Protein Analysis
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After electrophoresis, the proteins were blotted into polyvinylidene difluoride membranes under semi-dry transfer conditions (Thermo Scientific, Rockford, IL). After blocking with 5% milk, the following antibodies were used: anti-VDR (1:200, sc-13133; Santa Cruz Biotechnology, Dallas, TX), anti-TNF-α (1:500, ab183896; Abcam, Cambridge, United Kingdom), and secondary antibodies (Code: 115-035-146; Code: 111-035-003, Jackson ImmunoResearch, West Grove, PA). Protein loading was normalized to anti-GAPDH (1:5,000, sc-365062; Santa Cruz). Bands were visualized by chemiluminescence detection (Chemiluminescent HRP Substrate, Millipore, MA) and quantified using the MicroChemi 2.0 System. Specific bands were quantified by densitometric analyses with GelQuant software (Jerusalem, Israel).
9
Western Blot and qRT-PCR Analysis
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Cellular or tissue proteins were extracted and analyzed by western blot. The total cell lysates were extracted from the cells or tissues, adding phosphatase and protease inhibitors. Total proteins (20 μg) were separated via 8–15% SDS-PAGE and transferred onto PVDF membrane (Millipore, USA). Next, the membranes were blocked with 5% BSA and incubated with primary antibodies (dilution in 5% BSA-TBST) for overnight at 4 °C. Then, probed it with secondary antibody for 1 h at room temperature. Subsequently, the expression of the target proteins was detected by Chemiluminescent HRP Substrate (Millipore, USA). The antibodies used in this study were presented in Additional file 1 .
The total cellular RNA was isolated using the TRIzol Reagent (Vazyme, Nanjing, China) and reverse transcribed with the HiScript QRT SuperMix for qPCR (Vazyme). The mRNA levels were measured using the SYBR Green master mix (Vazyme), the primers for qPCR assay were showed in Additional file2 : Tables S1 and S2.
The total cellular RNA was isolated using the TRIzol Reagent (Vazyme, Nanjing, China) and reverse transcribed with the HiScript QRT SuperMix for qPCR (Vazyme). The mRNA levels were measured using the SYBR Green master mix (Vazyme), the primers for qPCR assay were showed in Additional file
10
HOTAIR RNA-Binding Assay with EZH2
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Cell nuclear extracts were applied to RNA-binding assays using the Nuclear and Cytoplasmic Extraction Kit according to the manufacturer’s protocol. Biotin-labeled wild-type HOTAIR oligonucleotides (5′-GUGGGGAGUGGAGAGAGGGAGCCCA-3′) or mutated HOTAIR oligonucleotides (HOTAIR-36U46A: 5′-GUGGGUAGUGGAGAGAGGGAGCCCA-3′; HOTAIR 36G46G: 5′-GUGGGGAGUGGAGAGGGGGAGCCCA-3′; HOTAIR 36U46G: 5′-GUGGGUAGUGGAGAGGGGGAGCCCA-3′) were used for the EMSA. The reaction mixture was loaded on a 5% native polyacrylamide gel in 0.5% Tris-borate-EDTA and blotted onto membranes (Millipore). After 7 min of UV cross-linking, the interaction between the biotin-labeled HOTAIR oligonucleotides and the EZH2 protein was detected using a chemiluminescent EMSA kit (Beyotime) with a streptavidin-HRP conjugate and a chemiluminescent HRP substrate (Millipore).
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