For Western blots, proteins (20 μg per lane) were separated by SDS-PAGE and transferred to a PVDF membrane (Millipore). Membranes were blocked in 4% milk in TBST (0.05M Tris pH7.2, 0.15M NaCl, 0.1% Tween20) for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C or for 1 hour at room temperature. Primary antibodies and dilutions used for western blots were Fyn (clone 59, BioLegend, 1:1000), GluA1 (1504, Millipore, 0.001 mg/ml), Homer1 (AT1F3, LSBio, 1:1000), mGluR5 (5675, Millipore, 1:2000), PSD95 (K28/43, BioLegend, 1:500), beta-Actin (GTX109639, GeneTex, 1:10,000), p-ERK T202/Y204 (4370, Cell Signaling Technologies, 1:1000), and total ERK (9102, Cell Signaling Technologies, 1:1000). Primary antibodies were detected using species-specific HRP-conjugated secondary antibodies. Blots were developed using Femto Maximum Sensitivity Substrate (Pierce) and imaged using a ProteinSimple imaging system (San Jose, CA).
Beta actin
Manufactured by GeneTex
Sourced in United States
Beta-actin is a highly conserved cytoskeletal protein that is ubiquitously expressed in all eukaryotic cells. It is a component of the microfilament system and plays a crucial role in maintaining the structural integrity of cells and facilitating cellular processes such as cell motility, cytokinesis, and organelle movement.
Automatically generated - may contain errors
Lab products found in correlation
P erk1 2, by Cell Signaling Technology (2 mentions)
Histone h3, by Abcam (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Total erk, by Cell Signaling Technology (1 mentions)
Glua1, by Merck Group (1 mentions)
Perk t202 y204, by Cell Signaling Technology (1 mentions)
Mglur5, by Merck Group (1 mentions)
Cyclin d1, by GeneTex (1 mentions)
Erk1 2, by GeneTex (1 mentions)
Foxo3, by GeneTex (1 mentions)
Cyclin a2, by GeneTex (1 mentions)
Cyclin b1, by GeneTex (1 mentions)
Phosphosafe extraction reagent, by Merck Group (1 mentions)
Phospho c jun, by Abcam (1 mentions)
Lamin a c, by Abcam (1 mentions)
Phospho c fos, by Cell Signaling Technology (1 mentions)
Malt1, by Santa Cruz Biotechnology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Cleaved parp, by GeneTex (1 mentions)
Cleaved caspase 3, by GeneTex (1 mentions)
15 protocols using beta actin
1
Western Blot Protein Analysis Protocol
2
Lung Cancer Cell Culture and Antibody Characterization
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Lung adenocarcinoma cells HCC827, H1975, H1650, and A549 and nonmalignant lung cells 16HBE were purchased from Nanjing Cobioer Co., Ltd. HCC827, H1975, and H1650 were cultured in RPMI-1640 medium enriched with 10% FBS and allowed to grow under 37°C and 5% CO2 conditions. However, A549 and 16HBE were cultured in F12K and DMEM/high glucose medium enriched with 10% FBS under 37°C and 5% CO2 conditions. We purchased the antibodies of NMU (Affinity, Cat No. DF4238), p-Erk1/2 (Cell signaling technology (CST), Cat No. 4370), Erk1/2 (GeneTex, Cat No. GTX59618), p-FoxO3 (CST, Cat No. 13129), FoxO3 (CST, Cat No. 2493), cyclin A2 (CST, Cat No. 4656), cyclin B1 (CST, Cat No. 12231), cyclin D1 (CST, Cat No. 2922), P21 (CST, Cat No. 2947), P27 (CST, Cat No. 3686), and beta-actin (CST, Cat No. 58169).
3
Western Blotting Analysis of Cellular Proteins
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For Western blotting analysis, the cells (60–80% confluency) were washed twice by PBS and scraped. The cells (1 × 106 cells) were disrupted by the lysis buffer (10 mM Tris-HCl, 5 mM EDTA, pH 8.0, phosphatase inhibitors, and protease inhibitors) and kept on ice for 30 min. The protein mixture was subjected to SDS-PAGE and transferred onto a PVDF membrane followed by blocking with 5% (w/v) skim milk. The membrane was then incubated with primary antibodies (1:1000 in 5% skim milk in TBST) for 2 h at room temperature and horseradish peroxidase-conjugated secondary antibody (1:10,000) for 1 h at room temperature followed by enhanced chemiluminescent (EMD Millipore Co., Billerica, MA, USA) detection. The primary antibody against cleaved PARP (~89 kDa) was purchased from Cell Signaling Inc., Danvers, MA, USA. The primary antibodies against beta-actin (~42 kDa), TP53 (~53 kDa), XAF1 (~38 kDa), and MDM2 (~90 kDa) were purchased from GeneTex Inc., Hsinchu City, Taiwan. The experiments were repeated at least twice, and representative figures are shown.
4
Immunoblotting analysis of NF-κB signaling
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Cells were lysed in PhosphoSafe™ Extraction Reagent (Merck Millipore, MA, USA) and boiled in TOOLS SDS-PAGE loading buffer (BIOTOOLS, Taipei, Taiwan). Cell lysates were analyzed on 10% sodium dodecyl sulfate polyacrylamide gel. Proteins were detected by immunoblotting using primary antibodies against MALT1 (Santa Cruz, TX, USA), Cylindromatosis 1 (CYLD) (Santa Cruz), regnase-1 (gift of Dr. Shizuo Akira) (25 (link)), RelB (Cell signaling, MA, USA), p50 (Santa Cruz), p65 (Santa Cruz), phospho-c-Jun (Abcam), phospho-c-Fos (Cell signaling), lamin A+C (Abcam, MA, USA), and beta-actin (GeneTex, Hsinchu, Taiwan). Horseradish peroxidase-conjugated goat anti-rabbit (GeneTex) and rabbit anti-mouse (GeneTex) antibodies were used as secondary antibodies. The intensity of the blots was quantified by ImageJ™ software (NIH, USA).
5
Signaling Pathway Analysis of DOK Cells
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First, 6 × 105 DOK
cells were seeded into 6 cm plates and incubated overnight. Following
the treatment of DOK cells with BAK, Stattic, or Wortmannin, cell
lysates were obtained and lysed in an RIPA buffer containing a proteinase
inhibitor. The protein lysates were loaded onto SDS-PAGE, and proteins
were transferred onto PVDF membranes and reacted with primary antibodies
followed by secondary antibodies (Jackson, USA). Finally, the signals
were amplified by ECL (Perkin Elmer, USA), imaged by ChemiDoc XRS+
System (BioRad, USA) and quantified by Image Lab software (BioRad,
USA). Primary antibodies against pSTAT3(Tyr705), STAT3, pAkt(Ser473),
Akt, cleaved caspase-3, cleaved caspase-9, cleaved caspase-7, cleaved
PARP, γH2AX, and beta-Actin (GeneTex, USA) were used for Western
blotting.
cells were seeded into 6 cm plates and incubated overnight. Following
the treatment of DOK cells with BAK, Stattic, or Wortmannin, cell
lysates were obtained and lysed in an RIPA buffer containing a proteinase
inhibitor. The protein lysates were loaded onto SDS-PAGE, and proteins
were transferred onto PVDF membranes and reacted with primary antibodies
followed by secondary antibodies (Jackson, USA). Finally, the signals
were amplified by ECL (Perkin Elmer, USA), imaged by ChemiDoc XRS+
System (BioRad, USA) and quantified by Image Lab software (BioRad,
USA). Primary antibodies against pSTAT3(Tyr705), STAT3, pAkt(Ser473),
Akt, cleaved caspase-3, cleaved caspase-9, cleaved caspase-7, cleaved
PARP, γH2AX, and beta-Actin (GeneTex, USA) were used for Western
blotting.
6
Antibodies for Epithelial-Mesenchymal Transition
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Mouse monoclonal antibodies (mAbs) against human E-cadherin (clone 36, BD Biosciences, San Jose, CA), human Snail1 (clone L7042, Cell Signaling, Danvers, MA), human vimentin (clone V9, ZYMED, Carlsbad, CA), human vinculin (clone hVIN-1, Sigma, St. Louis, MO), human vitronectin (clone 342603, R&D systems, Minneapolis, MN), human fibronectin (clone 10/Fibronectin, BD Biosciences, San Jose, CA), as well as polyclonal antibodies (pABs) against human claudin-1 (clone MH25, Invitrogen, Waltham, MA), human occludin (clone Z-T22, ZYMED, Carlsbad, CA), human integrin alpha 5 (Ag0860, catalog number 0569-1-AP, Proteintech, Chicago, IL), beta-actin (catalog number GTX109639, Genetex, Irvine, CA), and alpha tubulin 4a (catalog number GTX112141, Genetex, Irvine, CA) were purchased as indicated. Anti-mouse and anti-rabbit IgG horseradish peroxidase conjugates (Jackson Immunoresearch Laborattories, Inc, West Grove, PA), rhodamine X-conjugated phalloidin (Wako, Osaka) and thiazolyl blue tetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO) were also purchased as indicated.
7
Western Blotting of Cellular Proteins
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In six-well plates, 3.5 × 105 cells/well incubated at 37°C for overnight attachment prior to any treatment. Proteins were extracted from cells in 2x Laemmli buffer (Bio-Rad) containing protease and phosphatase inhibitors. Except as noted, equal sample volumes were applied to SDS-PAGE gels and transferred to PVDF membranes. Membranes were incubated in Odyssey Blocking Buffer (Li-Cor) overnight at 4°C prior to probing with primary antibodies overnight at 4°C. IRDye rabbit, mouse, and goat secondary antibodies (Li-Cor) were applied to probed membranes for 45 m at room temperature, and visualized using a Li-Cor Odyssey scanning system. Primary antibodies used for standard immunoblot analysis included AXL (R&D Systems), pAktS473 and pan-Akt (Cell Signaling Technology), and beta-actin (Sigma). Based on minimal detection by standard methods, pAXLY702 (Cell Signaling Technology), GAS6 (GeneTex), and beta-actin (as system control) antibodies were used to detect corresponding protein levels by Wes capillary electrophoresis (ProteinSimple) based on the manufacturer's protocol. Chemiluminescent signal is depicted as traditional immunoblot protein bands, which are generated by Compass software (ProteinSimple), as done previously for RTKs (Furugaki et al., 2016 (link)).
8
Quantifying PRLR Protein in Xenograft Tumors
9
Chromatin Immunoprecipitation of β-Catenin in Xenopus
10
Cofilin Phosphorylation Assay with IL-10 Treatment
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