106 HMC-1 cells in 1 ml were treated with 20 μg/ml E4 or A1 antibody for 15 min at 37 °C, followed by 100 ng/ml SCF for 3 min at 37 °C; 30 mg/lane of protein extract was resolved in 4–12 % gradient SDS-PAGE gel transferred on 2 blots, probed with either pY99 [Santa Cruz, sc-7020], anti-Kit E1 [Santa Cruz, sc-17806] and beta-actin C4 [Santa Cruz, sc-47778].
β actin c4
Manufactured by Santa Cruz Biotechnology
Sourced in United States, Canada
β-actin (C4) is a monoclonal antibody that recognizes the β-actin protein. β-actin is a ubiquitously expressed cytoskeletal protein involved in various cellular processes. The C4 clone of the β-actin antibody is a useful tool for the detection and quantification of β-actin in a variety of applications, such as Western blotting, immunohistochemistry, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Bcl 2, by Cell Signaling Technology (5 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Cyclin d1, by Santa Cruz Biotechnology (4 mentions)
Gel imaging system, by Bio-Rad (4 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Survivin, by Cell Signaling Technology (3 mentions)
Whatman, by Cytiva (3 mentions)
Caspase 3, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Infrared dye conjugated secondary antibody, by LI COR (2 mentions)
Ar d6f11, by Cell Signaling Technology (2 mentions)
Prostease inhibitor cocktail, by Roche (2 mentions)
Foxa1 ab23738, by Abcam (2 mentions)
Hoxb13 f 9, by Santa Cruz Biotechnology (2 mentions)
Flag m2, by Merck Group (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
α tubulin b 7, by Santa Cruz Biotechnology (2 mentions)
Protein assay kit, by Bio-Rad (2 mentions)
82 protocols using β actin c4
1
Antibody-Mediated SCF Signaling in HMC-1 Cells
2
Western Blot Analysis of Nrf2, HO-1, and Apoptosis
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Cells were lysed in passive lysis buffer (Promega) and harvested with a cell scraper. Cell debris was removed by centrifuging the cell lysate at 13,000 rpm for 10 minutes at 4°C, and 30 μg of proteins were loaded on 10% SDS-PAGE gels and separated by gel electrophoresis. Proteins were then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and blocked for 1 h with 10% nonfat milk in Tris-buffered saline with 0.1% Tween 20. Proteins were then blotted with antibodies against Nrf2 (C-20, Santa Cruz Biotechnology, Santa Cruz, CA), HO-1 (H-105, Santa Cruz Biotechnology), lamin B1 (A-11, Santa Cruz Biotechnology), cleaved caspase-3 (9661, Cell Signaling, Beverly, MA), and beta-actin (C4, Santa Cruz Biotechnology). Detection of the primary antibody was accomplished using HRP-conjugated anti-mouse IgG (1 : 3000, Santa Cruz Biotechnology) and anti-rabbit IgG (1 : 3000, Santa Cruz Biotechnology). Intensities of the protein bands were evaluated by densitometric analysis using GeneGnome XRQ (Syngene Corp., Cambridge, UK).
3
Protein Extraction and Immunoblotting Protocol
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Whole cell extracts were prepared by lysis in RIPA buffer (50 mM Tris pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with prostease inhibitor cocktail (Roche). Nuclear extracts were prepared by hypotonic lysis (10 mM HEPES pH 8.0, 5 mM MgCl, 250 mM Sucrose, protease inhibitors) followed by extraction with RIPA buffer. Immunoblotting was performed according to standard procedures using the following antibodies: AR D6F11 (Cell Signaling), HOXB13 F-9 (Santa Cruz), FOXA1 ab23738 (Abcam), beta-actin C4 (Santa Cruz). The Odyssey imaging system was used to quantitate fluorescent intensity of infrared dye conjugated secondary antibodies (LI-COR).
4
Protein Extraction and Immunoblotting Protocol
5
Comprehensive Western Blot Analysis of EVs
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For the Western blot analysis, EVs or
parental cells were lysed by RIPA buffer (1× for cells, 1:1 in
PBS for EVs) supplemented with a proteinase inhibitor cocktail. Proteins
were separated on ExpressPlus PAGE Ready gels 4–20% (A2S, M42015)
and transferred on the cellulose membrane (300 mA, 90 min) using a
BioRad blotting device (BioRad, USA). After overnight blocking in
5% milk in TBST, specific antibodies were applied to the membranes
for 1 h to detect markers: CD81 (B–II, Santa Cruz, USA); c-myc
(9E10, Santa Cruz, USA) (all 1:500); and beta-actin (C4, Santa Cruz,
USA) (1:1000). HRP-conjugated antimouse secondary HRP goat antimouse
IgG antibody (no. 4053, Biolegend, USA) (1:5000 in TBST) was applied
for 1 h before imaging using enhanced chemiluminescence substrate
EZ-ECL Kit (Biological Industries, catalog no. 20-500-120). An ALFA
tag was detected by a homemade fluorescent anti-ALFA tag nanobody
and visualized by a fluorescent reader at 642/662 nm wavelength.
parental cells were lysed by RIPA buffer (1× for cells, 1:1 in
PBS for EVs) supplemented with a proteinase inhibitor cocktail. Proteins
were separated on ExpressPlus PAGE Ready gels 4–20% (A2S, M42015)
and transferred on the cellulose membrane (300 mA, 90 min) using a
BioRad blotting device (BioRad, USA). After overnight blocking in
5% milk in TBST, specific antibodies were applied to the membranes
for 1 h to detect markers: CD81 (B–II, Santa Cruz, USA); c-myc
(9E10, Santa Cruz, USA) (all 1:500); and beta-actin (C4, Santa Cruz,
USA) (1:1000). HRP-conjugated antimouse secondary HRP goat antimouse
IgG antibody (no. 4053, Biolegend, USA) (1:5000 in TBST) was applied
for 1 h before imaging using enhanced chemiluminescence substrate
EZ-ECL Kit (Biological Industries, catalog no. 20-500-120). An ALFA
tag was detected by a homemade fluorescent anti-ALFA tag nanobody
and visualized by a fluorescent reader at 642/662 nm wavelength.
6
Immunoblot Detection of RNase Proteins
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Cells were resuspended in native lysis buffer supplemented with cOmplete Protease Inhibitor and incubated on ice for 30 min. Lysates were cleared by centrifugation (10,000 rcf, 5 min, 4 C). To deglycolysate proteins, cleared lysates and purified RNases were denatured and treated with PNGaseF (New England Biolabs, Frankfurt a.M., Germany) according to manufacturer's recommendations. RNaseT2 and RNase2 were detected with primary antibodies ab140191 (Abcam, Berlin, Germany; 1:1000)) and PA5-97305 (Thermo Fisher Scientific; 1:500), respectively. For standard immunoblot, secondary goat-anti-rabbit-HRP (Bio-Rad Laboratories, Feldkirchen, Germany; 1:5000) and Pierce ECL Subtrate (Thermo Scientific) were used, for quantitative immunoblot, secondary goat anti-rabbit IRDye800CW (Li-Cor Biosciences, Bad Homburg, Germany; 1:15,000) was used. Beta-actin (C4, Santa Cruz Biotechnology, Heidelberg, Germany; 1:1000), Flag (M2, Sigma-Aldrich; 1:1000) and HA (26183, Thermo Fisher Scientific; 1:1000) were detected with HRP-coupled primary antibodies and ECL substrate. Blots were recorded on an Odyssey FC Dual imaging system (Li-Cor Biosciences) .
Oligonucleotides RNA oligonucleotides were ordered from Biomers, Ulm, Germany and from Integrated DNA Technologies (IDT), Leuven, Belgium. DNA oligonucleotides were ordered from Integrated DNA Technologies (IDT), Leuven, Belgium.
Oligonucleotides RNA oligonucleotides were ordered from Biomers, Ulm, Germany and from Integrated DNA Technologies (IDT), Leuven, Belgium. DNA oligonucleotides were ordered from Integrated DNA Technologies (IDT), Leuven, Belgium.
7
Phosphoproteomic Analysis of STAT3
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The cells were lysed using 50 μL of RIPA lysis buffer per 1 million cells. SDS-PAGE gels (10%) were loaded with 30 μg of protein for separation and then transferred to a nitrocellulose membrane using a Trans-Blot Turbo System (Bio-Rad). The membrane was blocked in TBST with 5% nonfat skimmed milk for 1 hour at room temperature. Blots were probed for pSTAT3-Y705 (D3A7 XP, Cell Signaling Technology) and reprobed for tSTAT3 (F-2, Santa Cruz Biotechnology Inc.). The images were taken using a GBox imager. Densitometry analysis of the blots was carried using ImageJ (NIH) normalized to the housekeeping gene B-Actin (C4, Santa Cruz Biotechnology Inc.).
8
Assessing Immunoregulatory Molecules in Lung Lysates
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20 to 40 µg of total lung lysates were prepared with RIPA lysis buffer (ThermoFisher Scientific, Waltham, MA, USA) and subjected to the evaluations according to the procedures that we recently reported (30 (link)). Anti-mouse ICOS (7E.17G9, Bio X Cell) and anti-mouse ICOSL (HK5.3, Bio X Cell), anti-mouse CD28 (37.51, Bio X Cell), anti-human-CTLA-4 (922101, R&D), anti-mouse-CTLA-4 (a generous gift from Prof. Lieping Chen, Medical Oncology, Yale School of Medicine), anti-mouse B7-1 (F-7, Santa Cruz Biotechnology) and anti-mouse B7-2 (BU63, Santa Cruz Biotechnology) and b-actin (C4, Santa Cruz Biotechnology) were used as primary antibodies and immunoreactive bands were captured and analyzed using a ChemiDoc (Bio-Rad) imaging system.
9
Antibody Detection Protocol for Cells
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Antibodies used for immunofluorescence and western blotting included NQO1 (A180, Santa Cruz, La Jolla, CA), PARP1 (F-2, Santa Cruz), β-actin (C4, Santa Cruz), α-tubulin (B-7, Santa Cruz), PAR (Trevigen, Gaithersburg, MD), γH2AX (JBW301, Millipore, Temecula, CA), H2AX (938CT5.1.1, Cell Signaling, Danvers, MA), and catalase (12980S, Cell Signaling, MA).
10
SIV Gag Protein Detection Protocol
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For SIV Gag Western blot analysis, TeloRF cell lysates were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) transfer membranes (Amersham) using standard laboratory procedures. The membranes were blocked for nonspecific protein binding overnight at room temperature in 5% skim milk and 0.1% Tween20 in PBS and then probed using SIV Gag p27 monoclonal antibody [42 (link)]. An anti-mouse IgG peroxidase conjugated antibody (Sigma clone: A5906) and 3,3’-diaminobenzidine (DAB; Vector Laboratories) were used for immunodetection. Western blot analysis for β-Actin (C4) (Santa Cruz Biotechnology; sc-47778 HRP) was also performed on the TeloRF cell lysates according to the manufacturer’s instructions.
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