Cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail tablets (Roche). Equivalent amounts of cell extracts were separated by 10% SDS-PAGE and transferred onto PVDF. Filters were incubated with anti-CXCR4 antibody (1：1,000) at 4℃ overnight. Then, the secondary antibodies were added at a 1 : 5,000 (Thermo, MA, USA) dilution at room temperature for 1 h and detected by chemiluminescence using ECL Hyperfilm.
Hyperfilm ecl
Manufactured by Thermo Fisher Scientific
Sourced in United States
Hyperfilm ECL is a high-performance X-ray film designed for the detection and visualization of chemiluminescent signals in Western blotting and other applications. It provides high sensitivity and low background, allowing for the accurate detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Western ecl substrate, by Thermo Fisher Scientific (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Fluor s multimager, by Bio-Rad (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti jagged1, by Santa Cruz Biotechnology (1 mentions)
Anti notch1, by Santa Cruz Biotechnology (1 mentions)
Anti klf4, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Hrp conjugated secondary antibody, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Cleaved caspase 3 antibody, by Abcam (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
8 protocols using hyperfilm ecl
1
CXCR4 Protein Expression Analysis
2
Western Blot Analysis of STAT3 and Raptor
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Western blot analysis was performed according to published protocols [13] (link). The membrane was incubated at 4°C overnight with specific antibodies directed against STAT3 (1∶1000), pSTAT3 (1∶2000 diluted in distilled water with 5% BSA), Raptor (1∶500), p-Raptor (1∶500) or β-actin (1∶3000). After incubation with primary antibodies, the membrane was washed three times in TBS-0.1% Tween 20 for 15 min each before incubated with species specific secondary antibodies (1∶10000) for 1 h at room temperature. Secondary antibodies were diluted in TBS-0.1% Tween 20 with 5% milk if not differently indicated. To visualize the bands the membrane was incubated in clarity western ECL substrate (Thermo Fisher Scientific, Bonn, Germany) for 5 min for chemiluminescent detection and exposed to an ECL hyperfilm (Th. Geyer GmbH & Co. KG, Hamburg, Germany). For the analysis of β-actin, the membranes were stripped and reprobed with an anti β-actin antibody to account for protein loading variations. Relative density of bands was evaluated by densitometry with Image J software. The mean density of the untreated sample ( = control) bands was assigned an arbitrary value of 1, and the mean density of the treated groups are expressed relative to the control groups.
3
Western Blot Analysis of Protein Expression
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Total proteins were extracted from treated cells with cell lysis buffer (RIPA, Beyotime, Shanghai, China). Then, proteins were subjected to 10% SDS-PAGE and transferred to PVDF membranes (Sigma-Aldrich). The PVDF membranes were blocked in TBST buffer (TBS supplemented with 0.1% Tween-20) containing 5% (wt/vol) skimmed milk powder for 2 h at room temperature. Followed by three washes in TBST, the PVDF membranes were then incubated with primary antibodies, including anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-KLF4 (Santa Cruz Biotechnology), anti-Notch1 (Santa Cruz Biotechnology), and anti-Jagged1 (Santa Cruz Biotechnology) respectively overnight at 4℃. After being washed three times in TBST for 10 min, the PVDF membranes were further probed with HRP-conjugated secondary antibodies (Abcam, Cambridge, UK). Lastly, the membranes were exposed to Hyperfilm-ECL (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min, and visualized using a Fluor S Multimager and Quantity One 4.1 (Bio-Rad Laboratories).
4
Immunoblot Analysis of NF-κB Pathway Proteins
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Protein was isolated using cell lysis buffer containing 20 mM Tris (pH7.5), 150 mM NaCl, 2 mM EDTA, 1% TritonX-100 (pH7.1) and complete protease inhibitor cocktail (Sigma Aldrich). Polyvinylidene difluoride or nitrocellulose membranes were prepared according to standard methods. Primary antibodies used for immunoblot analysis of NF-κB pathways were: rabbit anti-human IKKα (2682), phospho-IKKα/β (2697), p100/p52 (4882), NIK (4994), RelB (clone C1E4), TRAF3 (4729), p65 (clone D14E12), p105/p50 (3035) and mouse anti-human IκBα (clone L35A5), all purchased from Cell Signaling and used at 1:1,000 dilution. For detection of tagged recombinant proteins, anti-human c-Myc (551101, BD Biosciences) was used at 1:1,000 dilution and horseradish peroxidase-coupled anti-HA (H6533, Sigma-Aldrich) was used at 1:3,000 dilution. For loading controls, mouse anti-human GAPDH (clone 6C5; Santa Cruz Biotechnology) and anti-human RCC1 (clone E-6; Santa Cruz Biotechnology) were used at 1:1,000 dilution. Horseradish peroxidase-conjugated goat anti-rabbit (Bio-rad) and goat anti-mouse (BD Biosciences) secondary antibodies were used at 1:10,000 or 1:50,000 dilution and detected using a chemiluminescent substrate (Amersham ECL Prime Western Blotting Detection Reagent, GE Life Sciences) together with Hyperfilm ECL (Fischer Scientific).
5
RGC Protein Extraction and Western Blot
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Cell protein extraction and Western blot analysis were performed as previously reported (Wu J. H. et al., 2015 (link); Gao et al., 2017 (link)). Briefly, cultured RGCs were lysed, and total proteins were extracted on ice with cell lysis buffer (Cell Signaling Technology, Boston, MA, USA) and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and then transferred to 0.22-μm PVDF membranes. After blocking with 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with primary antibodies against B cell lymphoma 2 (Bcl-2, Abcam, Cambridge, MA, USA), cleaved caspase-3 antibody (Abcam, Cambridge, MA, USA) and β-actin (Abcam, Cambridge, MA, USA). Signals were monitored by the Kodak Imaging System (Kodak 440CF) using ECL Western blot substrate (Hyperfilm ECL, Thermo Fisher Scientific, Rockford, IL, USA). Then, they were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).
6
Western Blot Analysis of PilE Protein Expression
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Whole-cell lysates were washed with ice-cold PBS and lysed in modified radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 25 mM HEPES, 2 mM EDTA, 1% [wt/vol] SDS) buffer containing a protease inhibitor cocktail (Fermentas GmbH). Equal amounts of whole-cell lysates were then boiled and analyzed by SDS-PAGE. After transfer on nitrocellulose (Optitran BA-S 83; Millipore), blots were probed with anti-PilE (SM1) (41 (link)) antibody or an anti Rmp4 and a secondary antimouse antibody coupled to horseradish peroxidase (HRP) (Cell signaling). Immunoblots were revealed by enhanced chemiluminescence (ECLplus and Hyperfilm ECL; Thermo Scientific and GE Healthcare Life Sciences, respectively). Expression of PilE was normalized to that of Rmp4, an inner membrane protein used as a loading control.
7
Dot Blot Assay for m6A Detection
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The dot blot assay was performed according to an online protocol (https://en.bio‐protocol.org/e2095 ). Briefly, RNA was isolated from cells as previously described and denatured at 95°C for 2 min. Then, 2000 ng of mRNA was spotted onto a Hybond‐N+ membrane (Cytiva, Shanghai, China) and crosslinked by UV (4000 × 100 μJ/cm2, twice; Spectronics, USA). The membrane was then washed and incubated in blocking buffer (5% skim milk, EpiZyme, Guangzhou, Guangdong, China) and then gently shaken overnight with an anti‐m6A antibody (synaptic system, 202003; Goettingen, Germany) at 4°C. The membrane was then washed, incubated with an anti‐mouse antibody (EpiZyme), and washed again. Finally, the membrane was exposed to Hyperfilm ECL (Thermo Fisher, Waltham, MA, USA), and images were acquired using a ChemiDoc™ imaging system (Bio‐Rad Laboratories, Inc., Hercules, CA, USA). Methylene blue (MB Solarbio) was used to interact with mRNA and as a loading control.
8
Western Blot Protein Extraction
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Tissues were frozen at -80°C immediately after dissection for protein extraction.
Then, they were suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and0.05% SDS, pH 7.4) while broken into pieces on ice. The sample buffer was added, and the supernatant was collected after centrifugation at 12,000 rpm for 10 min at 0°C. After boiling for 10 min, the denatured proteins were separated by polyacrylamide gel electrophoresis (PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane, which was purchased from Millipore (Billerica, MA, USA). The membranes were washed with methanol for 3min prior to staining. Then, the membranes were blocked with 5% low-fat dried milk in TBS containing 0.5% Tween-20 (TBS-T) prior to a 2-h incubation at room temperature with a 1:800 dilution of an anti-human p-STAT3 (Tyr 705) antibody (Cell Signaling Technology, Beverly, MA, USA). The anti-human β-actin antibody (bzbio.com) was used to indicate the amount of proteins loaded. After fully rinsing, the membranes were incubated with an HRP-conjugated secondary antibody (bzbio.com). The membranes were developed with HRP chemiluminescence detection reagent (Millipore), and then exposed to Hyper film ECL (Thermo Scientific, Waltham, MA, USA).
Then, they were suspended in RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and0.05% SDS, pH 7.4) while broken into pieces on ice. The sample buffer was added, and the supernatant was collected after centrifugation at 12,000 rpm for 10 min at 0°C. After boiling for 10 min, the denatured proteins were separated by polyacrylamide gel electrophoresis (PAGE), and then transferred to a polyvinylidene fluoride (PVDF) membrane, which was purchased from Millipore (Billerica, MA, USA). The membranes were washed with methanol for 3min prior to staining. Then, the membranes were blocked with 5% low-fat dried milk in TBS containing 0.5% Tween-20 (TBS-T) prior to a 2-h incubation at room temperature with a 1:800 dilution of an anti-human p-STAT3 (Tyr 705) antibody (Cell Signaling Technology, Beverly, MA, USA). The anti-human β-actin antibody (bzbio.com) was used to indicate the amount of proteins loaded. After fully rinsing, the membranes were incubated with an HRP-conjugated secondary antibody (bzbio.com). The membranes were developed with HRP chemiluminescence detection reagent (Millipore), and then exposed to Hyper film ECL (Thermo Scientific, Waltham, MA, USA).
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