Tissues were snap frozen in liquid nitrogen following dissection then thawed and homogenized in lysis buffer using a Polytron homogenizer (T10 basic ULTRA-TURRAX, IKA). Proteins were separated by SDS-PAGE and blotted onto PVDF membranes (Bio-Rad, Munich, Germany). Membranes were incubated with 1% blocking reagent (Roche, Mannheim, Germany) for 1 h, before incubating with primary antibody (1:1,000) diluted in 0.5% blocking solution overnight at 4 °C. After three washing steps with TBS-T the membranes were incubated with the respective secondary antibodies for 1 h at room temperature (peroxidase-coupled anti-rabbit A6154, Sigma–Aldrich, 1:2000, anti-mouse A4416, Sigma–Aldrich, 1:10,000). After three washing steps, the signals were visualized using Pierce ECL Western Blotting Substrate (Perbio Science, Bonn, Germany) and exposure to chemiluminescence film (Amersham, Braunschweig, Germany).
Anti mouse a4416
Manufactured by Merck Group
Sourced in United States
Anti-mouse A4416 is a laboratory product manufactured by Merck Group. It is a primary antibody that specifically binds to mouse immunoglobulins. The core function of this product is to facilitate the detection and identification of mouse-derived proteins or cells in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Phosphatase inhibitor, by Roche (2 mentions)
Chemiluminescence film, by Cytiva (1 mentions)
Anti rabbit a6154, by Merck Group (1 mentions)
Blocking reagent, by Roche (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ab24525, by Abcam (1 mentions)
Ab32575, by Abcam (1 mentions)
Mab1501r, by Merck Group (1 mentions)
Ab34710, by Abcam (1 mentions)
Anti rabbit a0545, by Merck Group (1 mentions)
Ecl reagent, by Cytiva (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Ab219649, by Abcam (1 mentions)
β actin actb, by Merck Group (1 mentions)
Acetyl nf κb p65 lys310, by Cell Signaling Technology (1 mentions)
Anti rabbit 7074, by Cell Signaling Technology (1 mentions)
8 protocols using anti mouse a4416
1
Western Blot Analysis of Protein Expression
2
Western Blot Analysis of Extracellular Matrix Proteins
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The expression of Collagen 1 a1 (Col1a1), Vimentin and alpha smooth muscle Actin α-SMA was evaluated by western blot. Cells were lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor (Roche)), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of proteins from cell extracts were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes (Millipore), therefore blocked (5% no fat milk in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 2% BSA; overnight at 4 °C or 2 h at room temperature): anti-α-SMA [E184] (ab32575, Abcam), anti-Vimentin (ab24525, Abcam), anti-Col1a1 (ab34710, Abcam) and anti-β-Actin (MAB1501R, Chemicon). After wash, the membranes were incubated with the appropriate HRP-conjugated secondary Ab (1:5000 in TBST + 2% BSA; 2 h at room temperature; anti-mouse A4416 and anti-rabbit A0545, Sigma). The immunoreactivity was detected by ECL reagents (Amersham), acquired with the ChemiDoc imaging system (Image Lab, Bio-Rad).
3
Anti-miR21 Effects on STAT3 Protein Expression
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LFs of BOS patiens teated with anti-miR21 for 48 h were collected for total protein extraction. Cells were washed with PBS, lysed with lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich, St. Louis, MI, USA) and phosphatase inhibitor (Roche), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Thirty micrograms of proteins from LFs extracts were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA) and, therefore, blocked (5% no fat milk in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 2% BSA; overnight at 4 °C or 2 h at room temperature): anti-STAT3 monoclonal antibody [9D8] (MA1-13042, Invitrogen, Carlsbad, CA, USA) and anti-β-Actin (MAB1501R, Chemicon, Kitakami, Japan). After washing, the membranes were incubated with the appropriate horseradish-peroxidase conjugated secondary Ab (1:5000 in TBST + 2% BSA; 2 h at room temperature; anti-mouse A4416, Sigma, Burlington, MA, USA). The immunoreactivity was detected by ECL reagents (Amersham, London, UK), acquired with the ChemiDoc imaging system (Image Lab, Bio-Rad, Berkeley, CA, USA).
4
Antibody Dilution Protocol for Protein Analysis
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Antibodies from Abcam were diluted as follows: recombinant anti-PIP5K1C (ab109192) 1:1000, PIP5K1B (ab154818) 1:1000, CHUK/IKK alpha 1:1000 and VCL/vinculin (ab219649) 1:10,000. Antibodies from Cell Signaling Technology were diluted as follows: PIP5K1A (9693) 1:1000 SQSTM1 (8025) 1:5000, LC3A/B (12,741) 1:5,000, p-RPS6 S240/244 (5364) 1:20,000, RPS6 (2217) 1:20,000, and HRP-conjugated secondary anti-rabbit (7074) 1:10,000. Antibodies from Sigma-Aldrich were PIP4K2C (SAB1407977) 1:10,000, ACTB/β-actin (A5441) 1:20,000, and anti-mouse (A4416) 1:10,000. Other antibodies were PIKFYVE (Millipore, MABS522) 1:1000
5
Western Blot Analysis of Liver Proteins
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Total liver tissue lysates were fractioned by 10% acrylamide gel. The proteins were then transferred to a nitrocellulose membrane and blocked in 5% milk protein diluted in TBST (0.1%) for 1 h. All primary Ab incubations were overnight: NF-κB p65 (8242s; Cell Signaling Technology), acetyl–NF-κB p65 (Lys310) (3045s; Cell Signaling Technology), β-actin (A5441; Sigma-Aldrich), acetyllysine (ab80178; Abcam), IKBα (4814S; Cell Signaling Technology), Sirt1 (07-131; MilliporeSigma), PPAR γ coactivator-1a1 (PGC-1α; NBP1-04676; Novus Biologicals), and PBEF (nicotinamide phosphoribosyltransferase [NAMPT]) (sc-393444; Santa Cruz Biotechnology). Next day, membranes were washed in TBST and incubated with secondary Abs (anti-mouse (A4416; Sigma-Aldrich) and anti-rabbit (7074S; Cell Signaling Technology) for 1 h. Proteins were detected with chemiluminescence (Amersham Bioscience).
6
PDHA1 Protein Expression Analysis
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Proteins were electrophoresed with 4%–20% SDS-PAGE gels and transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% BSA for 1 h at room temperature and incubated overnight at 4°C in primary antibody diluent. Then, the membrane was incubated with secondary antibody for 1 h at room temperature. All bands were measured and analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA). The primary antibody was anti-PDHA1 (1 µg/ml, A13687, ABclonal, CHN). The secondary antibodies such as horseradish peroxidase (HRP)-conjugated anti-rabbit (A6154) and anti-mouse (A4416) antibodies were from Sigma-Aldrich.
7
Cell Fractionation and Immunoblot Analysis
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The preparation of cell lysates and immunoblots analysis were performed via established protocols.56 (link), 57 (link) The Subcellular Protein Fractionation Kit for Cultured Cells from Thermo Scientific (78840; Waltham, MA, USA) was utilized to prepare fractions according to the manufacturer's instructions. Primary antibodies: anti-NDRG1 (HPA006881), anti-β-actin (A1978) from Sigma-Aldrich (St. Louis, MO, USA); anti-caveolin-1 (N20) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-vimentin (#5741), anti-E-cadherin (#3195), anti-β-catenin (9562), anti-snail (#3879), anti-slug (#9585), anti-ZEB1 (#3396), anti-TWIST1 (#46702), anti-Na-K-ATPase (#3010), anti-GAPDH (#2118), anti-Histone H2A (#3636) were from Cell Signaling Technology (Beverly, MA, USA). The secondary antibodies such as horseradish peroxidase (HRP)-conjugated anti-rabbit (A6154), anti-goat (A5420) and anti-mouse (A4416) antibodies were from Sigma-Aldrich.
8
Everolimus and PEG-LIP(ev)-HA400kDa Effect on mTOR Pathway
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LFs were seeded on 6-well plate at a density of 5 × 105 cells and after 24 h were incubated with PEG-LIP(ev), PEG-LIP(ev)-HA400kDa and everolimus alone, with the same concentration of everolimus (50 nM). After 24 h, cells were washed with PBS, lysed with lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 1% NP-40, protease inhibitor cocktail (Sigma Aldrich) and phosphatase inhibitor (Roche)), gently vortexed for 20 min at 4 °C and centrifuged for 15 min at 13,200 rpm at 4 °C. Supernatants were quantified by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Twenty micrograms of proteins were loaded and separated in 8% SDS-PAGE. After electrophoresis, the gels were transferred to PVDF membranes (Millipore), therefore blocked (5% BSA in 0.1% Tween 20 TBS) and incubated with the primary Ab (1:1000 in TBST + 5% BSA; overnight at 4 °C): anti-mTOR (1:1000) (PA1518—abcam), anti-p-mTOR(Ser2448) (1:1000) (PA585736—abcam), and anti-β-Actin (1:5000) (MA1-140—Thermo Fisher Scientific). After wash, the membranes were incubated with the appropriate horseradish-peroxidase conjugated secondary Ab (1:5000 in TBST + 5% BSA; 2 h at room temperature; anti-mouse A4416 and anti-rabbit A0545, Sigma). The immunoreactivity was detected by ECL reagents (BioRad, Segrate, Italy), acquired with the Uvitec alliance mini H9 (Uvitec Ltd, Cambridge, UK).
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