HCC1954 (ATCC), 786-0 (NCI-60), and ACHN (NCI-60) cells were cultured in RPMI-1640 media supplemented with 10% heat-inactivated FBS, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. HeLa cells (ATCC) were cultured in MEM supplemented with Earle's salts, 2 mM glutamine, 10% heat-inactivated FBS, 1.5 g/l sodium bicarbonate, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 50 U/ml of penicillin, and 50 μg/ml of streptomycin. The DDK inhibitor, PHA-767491, was synthesized as described previously [34] (link). Etoposide (#341205) was from EMD Millipore. The antibodies were purchased as indicated: CST: PARP (#9542), LATS1 (#3477), MST1 (#3682), MST2 (#3952), SAV1 (#13301), YAP/TAZ (#8418), pYAP S127 (#13008); Bethyl Laboratories Inc.: pMCM2 S53 (A300-756A), MCM2 (A300-122A), LATS2/LATS1 (A300-479A); MBL International Corporation: CDC7 (K0070-3S); Sigma: β-actin (A5441); GE Healthcare: anti-mouse-HRP (NA931V), and anti-rabbit-HRP (NA934V).
β actin
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
β-actin is a highly conserved cytoskeletal protein that is ubiquitously expressed in eukaryotic cells. It plays a crucial role in the maintenance of cell structure and is involved in various cellular processes, such as cell motility, division, and signal transduction.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (7 mentions)
β actin, by Cell Signaling Technology (5 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
Imagequant tl software, by GE Healthcare (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Imagequant las 4000 mini, by GE Healthcare (3 mentions)
Ecl plus kit, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Enhanced chemiluminescent detection kit, by GE Healthcare (2 mentions)
Imagequant 350, by GE Healthcare (2 mentions)
Hrp conjugated secondary antibodies, by Fujifilm (2 mentions)
Mouse anti β actin, by Merck Group (2 mentions)
Las4000 imager, by GE Healthcare (2 mentions)
Imagequant software, by GE Healthcare (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ecl detection system, by GE Healthcare (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lats1, by Fortis Life Sciences (1 mentions)
Anti mouse hrp na931v, by GE Healthcare (1 mentions)
Anti rabbit hrp na934v, by GE Healthcare (1 mentions)
32 protocols using β actin
1
Investigating Cell Proliferation Signaling
2
Western Blot Analysis of RCAN1 Protein
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According to the manufacturer's instructions, the total proteins were extracted using radio-immunoprecipitation analysis (RIPA) lysis buffer (Beyotime, Shanghai, China). After separation by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated overnight at 4°C with the following primary antibodies: rabbit anti-RCAN1 (Abcam, UK) and rabbit anti-β-actin (Abcam, UK). After the incubation with primary antibodies, the membrane was washed with 0.1% TBST for 3 times, 10 min for each time. The membrane was incubated at room temperature for 2 h with secondary antibody goat anti-rabbit IgG H&L (HRP) (Abcam, UK) and washed with PBST buffer 3 times, 10 min for each time. Protein expression was detected using an enhanced chemiluminescence kit (GE Healthcare, Chicago, IL, USA) with β-actin as an internal reference.
3
EMT Protein Expression Analysis
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Cells were lysed with RIPA buffer, protease inhibitor cocktail was added, and protein concentrations were determined using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA). Samples (80 μg/well) were subjected to SDS-PAGE and then transferred to PVDF membranes. Anti-MED30 (1:500, Protein Tech #16787-1-AP, Chicago, IL), anti-E-cadherin (1:1000, BD Bioscience #610181, San Jose, CA), anti-N-cadherin (1:1000, BD Bioscience #610920), anti-P-cadherin (1:1000, BD Bioscience #610227), anti-Twist1/2 (1:750, Santa Cruz Biotechnology #sc-15393, Santa Cruz, CA), anti-vimentin (1:1000, DAKO #M7020, Carpentaria, CA) and anti-β-actin (1:2000, Santa Cruz Biotechnology #sc-47778) antibodies were diluted in 5% skim milk and incubated with membranes at 4°C overnight. The appropriate secondary antibody was applied (1:2000, horseradish peroxidase-conjugated anti-mouse or anti-rabbit) at room temperature for 1 hr. Chemiluminescence detection (GE Health Care, Little Chalfont, United Kingdom) was used to visualize MED30, E-cadherin, N-cadherin, P-cadherin, Twist1, vimentin, and β-actin. Western blot analysis was performed in triplicate.
4
Western Blot Analysis of CD44v6, ESRP1, and PTBP1
5
Western Blot Analysis of Peri-Infarct Brain Tissue
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Brain tissue in the peri-infarct area was homogenized and sonicated in RIPA buffer (Thermo Scientific, Rockford, IL) containing phosphatase and protease inhibitors (Roche, Tokyo, Japan) and centrifuged. Total protein in the supernatant was measured with the BCA protein assay kit (Pierce, Rockford, IL). Protein was separated by 7.5 or 12% SDS-PAGE and transferred to a polyvinylidenedifluoride membrane. After blocking with 5% skim milk or BSA in Tris-buffered saline solution-Tween 20 (T-TBS), the membrane was incubated with the primary antibodies in Canget signal immunostain or T-TBS. The same primary antibodies as used for the immunohistochemical studies, rabbit polyclonal antibody against p-Akt (Cell Signaling Technology), MAP2, and mouse monoclonal anti-β-actin (Sigma, Tokyo Japan) were used. After incubation with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare, Buckinghamshire, UK), signals were detected by chemiluminescence using an ECL-plus kit (GE Healthcare). Images were analyzed with Image Quant LAS 4000 mini (GE Healthcare) and Image J software and quantified as the relative increase over the controls after normalization with β-actin.
6
Western Blot Analysis of Protease Inhibitors
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Proteins from cells lysed in RIPA buffer were separated using SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked using 5% skim milk in Tris-buffered saline containing Tween-20, incubated overnight at 4°C with primary antibodies directed against SerpinB2 (Abcam, Cambridge, UK), SerpinE1 (Abcam), uPA (Abcam), c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or phospho-c-Jun (Santa Cruz Biotechnology), and β-actin (Sigma-Aldrich, St. Louis, MA, USA), and then incubated with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology). Blotted membranes were visualized using enhanced chemiluminescence reagents (GE Healthcare, Piscataway, NJ, USA) and protein expression was depicted as intensity of each protein relative to that of β-actin using ImageJ software.
7
Western Blot Analysis of Protein Lysates
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Protein lysates were collected in RIPA buffer (50 mmol/l Tris pH 7.4, 1%
NP-40, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA),
1 mmol/l phenylmethylsulfonyl fluoride and protease inhibitor cocktail
(Roche), stored at −80 °C (30 min), defrosted on ice and
centrifuged at 4 °C (10 min,
11 269g ). The supernatant
was recovered and total protein concentration was assessed by Bio-Rad assay.
Total proteins (25 μg) were resolved on a 12% SDS–PAGE gel and
transferred onto a nitrocellulose membrane. Primary antibodies used were mouse
anti-p65 (Santa Cruz Sc-8008), mouse anti-β-actin (Sigma–Aldrich
A-5441), mouse anti-α-tubulin (Santa Cruz Sc-5286) and rabbit
anti-11β-HSD1 (Ricketts et
al. 1998 ).
Secondary antibodies (Dako, Ely, Cambridgeshire, UK) anti-mouse and anti-rabbit
conjugated with HRP were added at a dilution of 1/5000. Equal loading of protein
content was verified using β-actin and the bands were visualised using ECL
detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
NP-40, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EDTA),
1 mmol/l phenylmethylsulfonyl fluoride and protease inhibitor cocktail
(Roche), stored at −80 °C (30 min), defrosted on ice and
centrifuged at 4 °C (10 min,
11 269
was recovered and total protein concentration was assessed by Bio-Rad assay.
Total proteins (25 μg) were resolved on a 12% SDS–PAGE gel and
transferred onto a nitrocellulose membrane. Primary antibodies used were mouse
anti-p65 (Santa Cruz Sc-8008), mouse anti-β-actin (Sigma–Aldrich
A-5441), mouse anti-α-tubulin (Santa Cruz Sc-5286) and rabbit
anti-11β-HSD1 (
al. 1998
Secondary antibodies (Dako, Ely, Cambridgeshire, UK) anti-mouse and anti-rabbit
conjugated with HRP were added at a dilution of 1/5000. Equal loading of protein
content was verified using β-actin and the bands were visualised using ECL
detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK).
8
Protein Extraction and Western Blot Analysis
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The cell lysate was prepared using Complete Lysis-M (Roche, Basel, Switzerland) solution containing a cocktail of proteinase inhibitors and phosphatase inhibitors. The protein concentration was determined using the BCA protein assay (Pierce Chemical, Dallas, TX, USA). Samples were subjected to 4% to 20% Mini-PROTEAN TGX (BioRad, Hercules CA, USA) PAGE and transferred to PVDF membranes using Trans-Blot Turbo Transfer System (BioRad). The blots were probed with primary antibodies [STAT1 (CST, Cat: CST-14994, Danvers, MA, USA), phosphoSTAT1 (CST, Cat: CST-9167), Akt (CST, Cat: CST-9272), phosphoAkt (CST, Cat: CST-4060), p38α MAPK (CST, Cat: CST-9228), phospho-p38α MAPK (CST, Cat: CST-4511), phospho-mTOR (CST, Cat: CST-5536) and β-Actin (CST, Cat: CST-3700)] and then incubated with horseradish peroxidase-linked secondary anti-rabbit or anti-mouse antibodies (GE, Piscataway, NJ, USA). Original figures of western blot have been included as Supplementary Figure S5 and antibodies dilutions have been provided as Supplementary Table S1 .
9
Western Blot Analysis of miR-21a-5p
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Bone marrow-derived macrophages transfected with mmu-miR-21a-5p mimic or inhibitor were lysed with M-PER™ Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) supplemented with phosphatase and protease inhibitors (Roche). Equal amounts of proteins were separated on 12% SDS-PAGE gels and transferred to nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden). Membranes were blocked for 1 h in TBS with 0.1% Tween-20 containing 5% nonfat dry milk and incubated overnight with primary antibodies [anti-GBP5 dilution 1:500 (ProteinTech, Chicago, IL, USA) or β-actin (dilution 1:5,000, Cell Signaling Technology, Danvers, MA, USA)] at 4°C. Membranes were incubated with horseradish peroxidase-conjugated secondary antibody (dilution 1:1,000) and Luminol chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) was used for antibody detection. Densitometry analysis was performed using ImageQuant TL Software (GE Healthcare, Buckinghamshire, UK), and band intensities were normalized to β-actin.
10
Western Blot Analysis of Adipose Tissue Proteins
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Expression of HSL, its serine-563-phosphorylated form (HSL Ser563) and ATGL were measured in epididymal fat pad homogenates by western blot. Antibodies for immunoblotting were as follows: anti-HSL (1:2000, Cell Signaling Technology, USA), anti-HSL Ser563 (1:500, Cell Signaling Technology), anti-ATGL (1:2000, Cell Signaling Technology) and anti-β-actin (1:5000, Sigma). Epididymal adipose tissue homogenates (25 µg total protein per well) were separated by SDS page, and proteins transferred to a polyvinylidene fluoride membrane. Membranes were then blocked [5% milk and 0.1% TWEEN-20 in PBS (PBS-T)], and incubated at 4°C overnight in primary antibodies diluted in 1% BSA in PBS-T. After being incubated with HRP-conjugated secondary antibodies (1:500, Wako Chemicals) in 5% milk in PBS-T, blots were developed using an Enhanced Chemiluminescent Detection Kit (GE Healthcare). Digital images of protein bands were acquired (ImageQuant 350, GE Healthcare, UK) and semi-quantitative analysis of protein content performed by densitometry using ImageQuant TL software (GE Healthcare, UK), with values normalised to β-actin as a loading control.
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