Cell lysates were prepared using a lysis buffer (50 mM Tris-Cl pH 7.4, 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100) supplemented with protease inhibitors (Sigma-Aldrich). Subsequently, the proteins (50 μg) were separated by SDS-PAGE, transferred onto PVDF membranes (Bio-Rad; Hercules, CA, USA), and incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies (Bio-Rad). Protein bands were visualized using the Miracle-Star Western Blot Detection System (iNtRON Biotechnology, Seongnam, Korea). Antibodies against NRP2 (1:1,000 dilution, #sc-13117; Santa Cruz Biotechnology, Dallas, TX, USA), phospho-FAK (1:1,000 dilution, #sc-81493; Santa Cruz Biotechnology), FAK (1:1,000 dilution, #sc-557; Santa Cruz Biotechnology), phospho-MAPKAPK2 (1:1,000 dilution, #3041; Cell Signaling Technology, Danvers, MA, USA), MAPKAPK2 (1:1,000 dilution, #3042; Cell Signaling Technology), and β-actin (1:5,000 dilution, #BS6007M; Bioworld Technology, St. Louis Park, MN, USA) were used.
Phospho fak
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Phospho-FAK is a laboratory reagent used to detect and quantify the phosphorylated form of Focal Adhesion Kinase (FAK), a key signaling protein involved in cell adhesion and migration. This product allows researchers to analyze the activation and regulation of FAK in various cellular and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Santa Cruz Biotechnology (4 mentions)
Total akt, by Cell Signaling Technology (3 mentions)
Phospho akt, by Cell Signaling Technology (3 mentions)
Nitrocellulose membrane, by Bio-Rad (3 mentions)
Total erk1 2, by Cell Signaling Technology (3 mentions)
Phospho pi3k, by Cell Signaling Technology (3 mentions)
β actin, by Abcam (3 mentions)
Total fak, by Santa Cruz Biotechnology (3 mentions)
Total pi3k, by Cell Signaling Technology (3 mentions)
Goat anti mouse igg, by BD (3 mentions)
Phospho erk1 2, by Cell Signaling Technology (3 mentions)
Mmp 2, by Cell Signaling Technology (3 mentions)
Mmp 9, by Cell Signaling Technology (3 mentions)
Hrp conjugated goat anti rabbit igg, by BD (3 mentions)
Phospho src, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor, by Merck Group (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Phospho erk, by Santa Cruz Biotechnology (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
10 protocols using phospho fak
1
Western Blot Analysis of Signaling Proteins
2
Protein Expression Quantification by Western Blot
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Protein expression levels were determined by western blot analysis as previously described [71 (link)]. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations in the total cell lysates were measured by using bovine serum albumin as the standard. Samples containing equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), and proteins were then transferred onto nitrocellulose membranes (Bio–Rad Laboratories). Membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween 20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell Signaling #4022), MMP-9 (Cell Signaling #13667), total PI3K (Cell Signaling #4292), phospho-PI3K (Cell Signaling #4228), total Akt (Cell Signaling #4491), phospho-Akt (Cell Signaling #4060), total-ERK1/2 (Cell Signaling #9012), phospho-ERK1/2 (Cell Signaling #9101), total FAK (Santa Cruz, sc-558), phospho-FAK (Santa Cruz, sc-11765), or β-actin (Abcam, ab189073) overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, San Diego, CA, USA, 554021) and goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using ECL reagents.
3
CXCL1 Signaling Pathway Activation
4
Fibroblast Extracellular Matrix Regulation
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High-glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Rockville, MD, USA). Fibroblast growth supplement (FGS) was purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Antibiotic solutions (penicillin/streptomycin) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The Sirius Red/Fast Green Collagen Staining Kit was purchased from Chondrex (Woodinville, WA, USA). The NucleoSpin RNA Kit, RNA PCR Kit (Prime Script), and TB Green® Premix Ex Taq™ (Tli RNaseH Plus) solution were purchased from Takara Bio (Otsu, Japan). Enzyme-linked immunosorbent assay (ELISA; col1a1 and col3a1, FN, and elastin) kits were purchased from MyBioSource (San Diego, CA, USA).
Monoclonal IgG primary antibodies, including mouse anti-FAK, -phospho-FAK, and -GAPDH, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Appropriate biotin-conjugated goat anti-mouse IgG secondary antibodies were purchased from Abcam (Cambridge, UK). Small interfering ON-TARGETplus Human PTK2 siRNA and Dharma FECT 1 Transfection Reagent were purchased from Dharmacon (Lafayette, CO, USA).
The Sirius Red/Fast Green Collagen Staining Kit was purchased from Chondrex (Woodinville, WA, USA). The NucleoSpin RNA Kit, RNA PCR Kit (Prime Script), and TB Green® Premix Ex Taq™ (Tli RNaseH Plus) solution were purchased from Takara Bio (Otsu, Japan). Enzyme-linked immunosorbent assay (ELISA; col1a1 and col3a1, FN, and elastin) kits were purchased from MyBioSource (San Diego, CA, USA).
Monoclonal IgG primary antibodies, including mouse anti-FAK, -phospho-FAK, and -GAPDH, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Appropriate biotin-conjugated goat anti-mouse IgG secondary antibodies were purchased from Abcam (Cambridge, UK). Small interfering ON-TARGETplus Human PTK2 siRNA and Dharma FECT 1 Transfection Reagent were purchased from Dharmacon (Lafayette, CO, USA).
5
Western Blot Analysis of Protein Expression
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Protein expression levels were determined by western blot analysis as previously described [18 (link)]. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated via sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell Signaling #4022), MMP-9 (Cell Signaling #13667), total PI3K (Cell Signaling #4292), phospho-PI3K (Cell Signaling #4228), total Akt (Cell Signaling #4491), phospho-Akt (Cell Signaling #4060), total-ERK1/2 (Cell Signaling #9012), phospho-ERK1/2 (Cell Signaling #9101), total FAK (Santa Cruz, sc-558), phospho-FAK (Santa Cruz, sc-11765), or β-actin (Abcam, ab189073) overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, 554021) or goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using enhanced chemiluminescence (ECL) reagents.
6
Western Blot Analysis of Key Signaling Proteins
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The following primary antibodies were used for Western blot analysis: polycystin‐1 (sc‐130554, Santa Cruz Biotechnology), polycystin‐2 (sc‐10376, Santa Cruz Biotechnology), ERK1/2 (sc‐514302, Santa Cruz Biotechnology), phospho‐ERK1/2 (Abcam ab32538), mTOR (701483, Thermo Fisher Scientific), phospho‐mTOR (5536 CST), FAK (sc‐271126, Santa Cruz Biotechnology), phospho‐FAK (8556 CST), YAP (sc‐101199, Santa Cruz Biotechnology), TAZ (sc‐518026, Santa Cruz Biotechnology) and actin (MAB1501, Millipore). The following secondary antibodies were used: goat anti‐mouse IgG HRP‐conjugate (AP124P, Millipore), goat anti‐rabbit IgG HRP‐conjugate (AP132P, Millipore) and donkey anti‐goat IgG HRP‐conjugate (A00178, GenScript).
7
Western Blot Analysis of Stem Cell Signaling
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Total protein was extracted from MSCs using RIPA lysis buffer (Thermo Fisher Scientific). Cell lysates and tissue homogenates (30 μg protein) in sample buffer were separated by electrophoresis on an 8–12% sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane for probing with antibodies. After washing with Tris-buffered saline/Tween-20 buffer (0.05% Tween-20, 150 mM NaCl, 10 mM Tris-HCl; pH 7.6), membranes were blocked with 5% bovine serum albumin for 1 h at room temperature then incubated with primary antibodies against SMP30, p21, CDK2, CDK4, Cyclin E, Cyclin D1, phospho-FAK, phospho-Akt, TWIST, PrPC, c-Caspase3, β-actin, and GAPDH (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands were detected using enhanced chemiluminescence reagents (GE healthcare).
8
Western Blot Analysis of Protein Expression
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Protein expression levels were determined by western blot analysis, as previously described [61 (link)]. Cells were lysed in a buffer containing 50 mM Tris, 5 mM EDTA, 150 mM NaCl, 1 mM DTT, 0.01% NP 40, and 0.2 mM PMSF. The protein concentrations of the total cell lysates were measured by using bovine serum albumin as a standard. Samples containing equal amounts of protein were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose membranes (Bio-Rad Laboratories). The membranes were blocked with 5% skim milk in Tris-buffered saline containing Tween-20 at RT. Then, the membranes were incubated with primary antibodies against MMP-2 (Cell Signaling #4022), MMP-9 (Cell Signaling #13667), total PI3K (Cell Signaling #4292), phospho-PI3K (Cell Signaling #4228), total Akt (Cell Signaling #4491), phospho-Akt (Cell Signaling #4060), total-ERK1/2 (Cell Signaling #9012), phospho-ERK1/2 (Cell Signaling #9101), total FAK (Santa Cruz, sc-558), phospho-FAK (Santa Cruz, sc-11765), or β-actin (Abcam, ab189073) overnight at 4 °C and then with HRP-conjugated goat anti-rabbit IgG (BD Pharmingen, 554021) or goat anti-mouse IgG (BD Pharmingen, 554002) secondary antibodies for 60 min at RT. Antibody-bound proteins were detected using enhanced chemiluminescence (ECL) reagents.
9
Molecular Signaling Pathway Analysis
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The acarbose was purchased from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), glutamine, Penicillin-Streptomycin-Amphotericin B (PSA), fetal bovine serum (FBS), pyruvate and trypsin EDTA were purchased from Hyclone (Logan, UT, USA) and Gibco (Grand Island, NY, USA) for cell culture. The ammonium persulfate (APS), sodium dodecyl sulfate (SDS), bisacrylamide, TEMED, PVDF membranes, and NC membranes were purchased from Gibco (Grand Island, NY, USA) and Hyclone (Logan, UT, USA) for Western blot analysis. The antibodies to Ras, phospho-ERK, ERK, PCNA, PI3K, phospho-AKT, AKT, Rac1, RhoA, Cdc42, phospho-FAK, FAK, MMP9, MMP2, phospho-Src, Src and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Novus Biological (Littleton, CO, USA).
10
Western Blot Analysis of Cell Signaling
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Cells were lysed in ice-cold Laemmli sodium dodecyl sulfate (SDS) sample buffer. Equal amounts of proteins were separated by 10% or 12% SDS-PAGE and transferred onto PVDF membranes which were incubated with specific antibodies followed by horseradish peroxidase-labeled IgG. All antibodies were reacted with fibronectin, integrin α5, integrin β1, FAK, phospho-FAK (Tyr-397), ERK, phospho-ERK (Thr-202/Tyr-204), Akt, phospho-Akt (Ser-473), Src, phospho-Src (Tyr-416), cyclin D1, vimentin, Smad (Santa Cruz Biotechnology, Santa Cruz, CA, USA), phospho-Smad (Ser-423/425, Cell Signaling, Danvers, MA, USA), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (R&D Systems, Minneapolis, MN, USA). The signals in membranes were visualized using enhanced chemiluminescence (ECL) Western blotting reagents and the intensity of signals was determined by a computer image analysis system (Alpha Innotech Corporation, IS1000, San Leandro, CA, USA).
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