72 hours after transfection of si-VDAC1, A549 cells were collected and incubated in 200 μ L 1∗SDS-PAGE Sample Loading Buffer (Cat No. P0015L), 98°C for 10 minutes until the cells were completely lysis. Proteins were resolved on 10% gel using PAGE gel quick preparation kit (10%, CAT: PG112) and transferred to Trans-blot Turbo nitrocellulose membranes (Bio-Rad). Then, incubate the membrane in 10% milk and seal it for 2 hours. For primary antibody–protein hybridization, membranes were probed with the following antibodies at 4°C overnight (Beyotime): LDHA Rabbit Polyclonal Antibody, CAT: AF0216; COX IV Rabbit Polyclonal Antibody, CAT: AF6549; Hexokinase II Rabbit Polyclonal Antibody, CAT: AF7080; SDHB Rabbit Polyclonal Antibody, CAT: AF7956; and PGK1 Rabbit Monoclonal Antibody, CAT: AF1825. Thereafter, secondary anti-mouse (Beyotime CAT: LF101) or anti-rabbit IgG antibodies (Beyotime CAT: LF102) were incubated for 1 h at room temperature. Protein bands were developed with chemiluminescent reagents (Beyotime) and imaged with a Tanon 5200.
Trans blot turbo nitrocellulose membranes
Manufactured by Bio-Rad
Sourced in Spain, United States
The Trans-Blot Turbo nitrocellulose membranes are designed for efficient protein transfer from polyacrylamide gels to membranes for subsequent analysis. The membranes provide a high-binding capacity and uniform protein transfer.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (2 mentions)
β actin, by Merck Group (2 mentions)
Chemiluminescent reagent, by Beyotime (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Irdye 680lt goat anti mouse, by LI COR (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Sc 8312, by Santa Cruz Biotechnology (1 mentions)
T5168, by Merck Group (1 mentions)
Criterion tgx stain free gel, by Bio-Rad (1 mentions)
Anti rabbit igg hrp, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg hrp, by Thermo Fisher Scientific (1 mentions)
Chemismart 5000 system, by Vilber (1 mentions)
Anti gapdh, by Merck Group (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Trans blot turbo transfer pack, by Bio-Rad (1 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
10 protocols using trans blot turbo nitrocellulose membranes
1
VDAC1 Knockdown Protein Expression Analysis
2
Western Blot Analysis of Retinal CRX
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Whole retina protein lysates were prepared by homogenization of four genotype-matched isolated whole retinas from P10 mice and lysis in 1× RIPA buffer (Sigma) for 10 min with protease inhibitors (Aprotinin, Leupeptin, peptistatin, 0.1 mM Phenylmethaneslfonyl fluoride). Nuclear lysates were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagants (Thermo Scientific) with protease inhibitors. Either 30 µg of whole protein lysate or 5 µg of nuclear protein lysate was boiled for 10 min. Samples were run on a 4–11% SDS-PAGE gel and transferred onto Transblot Turbo nitrocellulose membranes (Bio-Rad) using the Transblot Turbo system (Bio-Rad). Membranes were probed with Rabbit anti-CRX 119b1 (1∶750) and Mouse anti-β-Actin (Sigma)(1∶1000). Goat anti-Mouse IRDye 680LT and Goat anti-Rabbit IRDye 800CW (LI-COR) were used as secondary antibodies. Signal was detected and quantified using the Odyssey Infrared Imager (LI-COR) and associated manufactory software. Kruskal-Wallis rank order test (Proc Npar1way of SAS, V9.3) was used to test for an overall difference among genotypes (p = 0.0002), then each genotype was compared to WT control (p<0.05). Post-hoc analyses were performeded using FDR p methods for multiple comparisons using PROC Multtest of SAS (V9.3) (FDR p<0.09) (n≥3).
3
Western Blot Analysis of Signaling Proteins
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After the experimental treatments, cells were lysed and solubilized in 200 μl of Laemmli loading buffer. Samples were boiled and sonicated for 5 min. Solubilized proteins (20 μl) were resolved by 8% and 10% SDS–PAGE and then electrophoretically transferred to 0.2 μm Trans‐Blot Turbo nitrocellulose membranes (BioRad, Alcobendas, Madrid). The membranes were blocked at room temperature for 90 min in Tris‐buffered saline containing 0.1% Tween 20 and 5% BSA. Then, the membranes were incubated overnight at 4°C with primary antibodies diluted in the same blocking solution. After that, the membranes were incubated with infrared dye‐conjugated antibodies (diluted 1:10,000; LI‐COR Biosciences, Lincoln, NE). Specific proteins were visualized by Odyssey Infrared Imaging System (LI‐COR Biosciences). The following primary antibodies were used: anti‐AKT (diluted 1:1000; SC‐8312, Santa Cruz), anti‐p‐AKT (diluted 1:1000; #9271, Cell Signaling), anti‐IGF‐1R (diluted 1:1000; #3027, Cell Signaling), anti‐p110α (diluted 1:1000; C73F8, Cell Signaling), anti‐p110β (diluted 1:1000; C33D4, Cell Signaling), anti‐p110δ (diluted 1:1000; D1Q7R, Cell Signaling) and anti‐α‐tubulin (diluted 1:5000; #T5168, Sigma‐Aldrich). Data were normalized to the α‐tubulin expression.
4
Western Blot Analysis of Neuronal Proteins
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Twenty-microgram proteins were separated using 4–20% CRITERION® TGX Stain-Free™ Gels (300 V, 20 min) and subsequently transferred to Trans-Blot® Turbo™ Nitrocellulose membranes (both Bio-Rad, Hercules, CA, USA). The membranes were blocked for 1 h at RT with 5% BSA in TBST (0.1% Tween20), before the primary antibodies were applied diluted 1:1000 in the same buffer (TrkA, Cell Signaling [2510]; pTrkA (Y674/675), Cell Signaling [4621S]; LMNA, Abcam [ab108595]; pLMNA (S22), Abcam [ab138450]; STMN1, abcam [ab52630]; pSTMN1 (S16), Abcam [ab47328]; pSTMN1 (S25), Abcam [ab194752]), except anti-Gapdh (EMD Millipore [MAB374]), which was diluted 1:2000. The membranes were incubated over night at 4 °C, washed with TBST and consecutively incubated with the appropriate secondary antibodies (anti-Rabbit IgG HRP, Thermo Scientific [31460]; anti-Mouse IgG HRP, Thermo Scientific [31430]) for 1 h at RT. Bands were visualized with SuperSignalTM Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and documented using a CHEMI SMART 5000 System (Vilber Lourmat, Eberhardzell, Germany).
5
Western Blot Analysis of Murine Pancreas
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Pancreas tissue (~30 mg) from 20 day old mice (prior to fat infiltration) was homogenized in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 mM Tris-HCl, pH7.5) using a polytron over ice. Insoluble components were pelleted by centrifugation (17,000 X g at 4°C). Equal amounts of protein (determined by Lowry assay, BioRad) in 2X Laemmli buffer were separated by 12% SDS–PAGE and either stained with silver salts or Coomassie brilliant blue, or blotted using the Trans–Blot Turbo Transfer Pack with the Trans-Blot Turbo Transfer System (BioRad). Trans-Blot Turbo nitrocellulose membranes (BioRad) were blocked in 5% (w/v) powdered skim milk (5% (w/v) goat serum for Novacastra CM5 p53 antibody) prior to overnight incubation with primary antibodies followed by species appropriate horseradish peroxidase-conjugated secondary antibodies (S4 Table ). Bound antibodies were visualized with Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) on the ChemiDoc MP Imaging System using Image-Lab 4.1 Software (BioRad).
6
Evaluating NF-κB Signaling Pathway
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Livers were homogenized in RIPA lysis buffer (EMD Millipore Corp, Temecula, CA, USA), containing 10 µL/mL of phosphatase I and II inhibitor and protease inhibitor (Sigma-Aldrich, St. Quentin Fallavier, France). Tissue homogenates were centrifuged for 15 min at 10,000 g, and supernatants were stored at −80 °C. Equal amount of proteins (40 µg) were run on a 4–15% gradient Mini-protean TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred onto Trans-Blot Turbo nitrocellulose membranes (Bio-Rad, Marnes-la-Coquette, France) and blotted with the following primary antibodies: phospho-IκBα (1/500), total-IκBα (1/500), Nuclear Factor kappa-B (NFκB)p65 (1/200) (all Santa Cruz Biotechnology, Heidelberg, Germany) and β-actin (1/5000, Sigma-Aldrich, St. Quentin Fallavier, France). Anti-mouse, anti-goat, or anti-rabbit IgG labeled with Dylight 800 or Dylight 680 was used as secondary antibodies. Proteins were determined by Infrared fluorescent detection (Odyssey, LI-COR Biosciences, Boulogne-Billancourt, France) and quantified using Image Studio Lab v5.2 software (LI-COR Biosciences, Boulogne-Billancourt, France).
7
Protein Extraction and Western Blot Analysis
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As previously described24 (link), protein was isolated from differentiated AEC cultures in situ from the transwell membrane surface using M-Per mammalian cell protein lysis reagent and Halt® protease and phosphatase inhibitor cocktail (both Thermo Scientific, Victoria, Australia). Protein samples were quantified using the BCA protein assay method (Bio-Rad, Victoria, Australia), and 10 μg electrophoresed using Novex® 4–12% gradient Bis–Tris denaturing gels (Life Technologies, Victoria, Australia) and electroblotted to Trans-Blot® Turbo nitrocellulose membranes (Bio-Rad). Membranes were blocked in 5% diploma skim milk and probed overnight at 4 °C with primary antibodies directed to Claudin-1 (37–4900), Occludin-1 (71–1500), ZO-1 (33–9100) (all Thermo Fisher Scientific, Waltham, MA, USA), or β-actin (A1978; Sigma-Aldrich Co., St Louis, MO, USA). Secondary antibody incubation was 1 h at RT with horse radish peroxidase-labelled secondary antibody (R&D Systems, MN, USA). Chemiluminescent imaging was performed using the LAS-3000 platform and histogram densitometry was performed using Multi Gauge software (V3.1 Fujifilm, Tokyo, Japan).
8
Western Blot Analysis of Ngn3 and Tubulin
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Cells were lysed and solubilized in 200 μL of Laemmli loading buffer. Samples were boiled and sonicated for 5 min. Solubilized proteins (20 μL) were resolved by 15% SDS–PAGE and then electrophoretically transferred to 0.2 μm Trans-Blot Turbo nitrocellulose membranes (BioRad, Alcobendas, Madrid). Membranes were blocked for 90 min at room temperature (RT) in Tris-buffered saline containing 0.1% Tween 20 and 5% BSA and then incubated overnight at 4°C with anti-Ngn3 rabbit polyclonal antibody (Abcam, Cat# ab38548, diluted 1:500) and anti-βIII tubulin mouse monoclonal antibody (TUJ1, dilution 1:1000 Covance, Emeryville, CA, United States) to ensure equal protein loading. Afterward, membranes were incubated for 1 h at RT with 1:10,000 infrared dye-conjugated secondary antibodies (LI-COR, United States), and proteins were visualized by Odyssey Infrared Imaging System (LI-COR). Densitometric analyses were performed using the ImageJ software (National Institutes of Health; freely available at https://imagej.nih.gov ). Data are presented as a Ngn3/βIII-tubulin ratio from 3 to 4 independent cultures.
9
Hepatic Protein Expression Analysis
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Frozen liver samples were homogenized on ice in Hepes lysis buffer (100 mg/mL) containing 75 mM NaCl, 750 μM magnesium chloride, 25 mM Hepes (pH 7.4), 500 μM EGTA, 5% glycerol, 0,5% Igepal, 1 mM dithiothreitol, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, and 1 mM sodium orthovanadate. A protease inhibitor cocktail (Sigma-Aldrich, St Louis, USA) was added before its use at a concentration of 5 μL/mL. All debris was removed through centrifugation at 15,000g at 4 °C for 15 min, and the supernatant obtained was used for analysis. Fifty micrograms of protein were separated in Criterion Gel 4–15% (BioRad, Hercules, USA) by electrophoresis and transferred to Trans-Blot Turbo Nitrocellulose membranes (BioRad, Hercules, USA). Western blotting and chemiluminescence detection using Luminata Clássico Western HRP Substrate (Millipore, Billerica, USA) were utilized to determine the catalytic subunit of glutamate cysteine ligase (GCLc), cystathionine gamma-lyase (CTH) and methionine adenosyltransferase 1A (MAT1A) and 2A (MAT2A) and GAPDH. The following antibodies were used: antibody against GCLc (1/1000) (Abcam, Cambridge, UK), antibody against CTH (1/500) (Abcam, Cambridge, UK), antibody against MAT1A (1/500) (Abcam, Cambridge, UK), antibody against MAT2A (1/500) (Abcam, Cambridge, UK) and antibody against GAPDH (1/1000) (Cell Signaling Technology, Danvers, USA).
10
Renal Protein Expression Analysis
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To study the expression of E-cadherin and α-smooth muscle actin, snap frozen renal cortex were lysed in Complete Lysis-M (04719956001; Roche). Protein concentrations were determined with the bicinchoninic acid method, and equal amounts of protein were resuspended in Laemmli sample buffer and boiled for 5 minutes at 95°C. The proteins were separated by SDS-PAGE on Criterion TGX gels (5671085; Bio-Rad) and transferred to TransBlot Turbo nitrocellulose membranes (1704159; Bio-Rad). The membranes were blocked for 1 hour in 20 mmol/L Tris, 150 mmol/L NaCl, and 1% Tween, pH 7.5/5% nonfat dry milk and incubated overnight at 4°C with the primary antibodies, including mouse monoclonal anti α-smooth muscle actin (ab7817; Abcam; 1/500 dilution), mouse monoclonal anti E-Cadherin (ab76055; Abcam; 1/1000 dilution), and mouse monoclonal anti β-actin (A2228; Sigma; 1/7500 dilution) antibodies. Subsequently, the membranes were washed and incubated with the corresponding secondary antibody, and immunoreactivity was visualized with enhanced chemiluminescence (ECL, RPN2106; GE Healthcare). ECL was detected on a Fujifilm LAS 4000. Digital pictures were quantified by densitometric analysis with ImageJ.
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