The gastrocnemius muscles of the ischemic limbs of mice were collected on the 3rd, 7th, and 28th days after ischemia of the hind limb and stored at - 80 °C. According to the manufacturer's instructions, the muscle tissue was homogenized in RIPA buffer (Beyotime, China) and centrifuged at 12000 rpm and 4 °C for 10 min. The protein supernatant was collected. The method described previously was used for western blot analysis [32 ]. The following antibodies were used: VEGF (Proteintech 1:1000 rabbit source), HIF-1α (affinity 1:1500 rabbit origin), MyoG (Abcam 1:1000 rabbit source), MyoD (Abcam 1:1000 rabbit source), goat anti-rabbit secondary antibody (Jackson 1:2000), and goat anti-mouse secondary antibody (Jackson 1:2000).
Goat anti mouse secondary antibody
Manufactured by Jackson ImmunoResearch
Sourced in United States
The Goat anti-mouse secondary antibody is a laboratory reagent used for the detection and quantification of mouse primary antibodies in various immunoassays and immunohistochemical applications. It is a polyclonal antibody produced by immunizing goats with mouse immunoglobulins, and then purifying the antibody fraction that specifically binds to mouse antibodies.
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Lab products found in correlation
Goat anti rabbit secondary antibody, by Jackson ImmunoResearch (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Vascular endothelial growth factor (vegf), by Proteintech (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Hif 1α, by Abcam (1 mentions)
Actin a 4700, by Merck Group (1 mentions)
Gammacell 1000, by Nordion (1 mentions)
3 3 diaminobenzidine (dab), by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
7500 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Abc elite reagent, by Vector Laboratories (1 mentions)
Superscript vilo, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Ab93926, by Abcam (1 mentions)
Ab13420, by Abcam (1 mentions)
Ab76956, by Abcam (1 mentions)
Ab38311, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Pierce universal nuclease for cell lysis, by Thermo Fisher Scientific (1 mentions)
Ab32572, by Abcam (1 mentions)
13 protocols using goat anti mouse secondary antibody
1
Muscle Tissue Analysis of Ischemic Limbs
2
Hypoxia and Radiation-Induced p53 Signaling
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A2780 cell lines were grown to 80% confluence in T150 flasks and exposed to hypoxic conditions using 5% O2 in a Bactron Anaerobic Chamber (Sheldon Manufacturing, Cornelius, OH, USA) for 8 or 24 h prior to harvesting. For radiation exposure, the A2780wtTP53 and A2780mTP53 cell lines were plated in 60 mm dishes, exposed to 5 Gy of ionizing radiation for 900 s using the Gammacell 1000 (MDS Nordion, Ottawa, ON, Canada) and harvested at 0, 2, 4, 6, 8, 24, and 48 h. Western blots were performed to evaluate p53 and p21 protein expression per established protocols (12 (link)). The following antibodies were used: p53 (sc-126, mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21 (AB-11, mouse monoclonal, NeoMarkers, Fremont, CA, USA), actin (A4700, mouse monoclonal, SIGMA, St. Louis, MO, USA), and goat anti-mouse secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA).
3
Curcumin Modulates VEGFR2 Phosphorylation
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Phosphorylation of VEGFR2 was investigated according to previously described [52 (link)]. Briefly, PAE-KDR cells were grown to 70–80% confluency in 6 well plates, starved for 16 hours in serum-free DMEM, and treated with various doses of curcumin for 8 hours, and stimulated with 100 ng/mL VEGF for 15 minutes. Cells were washed once with cold PBS and lysed on ice in 200 μL RIPA lysis solution supplemented with 1mM PMSF. Proteins were blotted onto nitrocellulose membranes and blocked overnight in 1% BSA in PBST, then incubated with a primary monoclonal mouse antibody against phosphotyrosine (Millipore; 05–321). Proteins were visualized using goat anti-mouse secondary antibody conjugated to HRP (Jackson ImmunoResearch; 715–035-150) and ECL detection system.
4
Girdin Protein Expression Analysis
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The 48 h after transfection, the cellular protein was extracted. After the protein concentration was measured via the BCA method, 5% stacking gel and 12% separating gel were performed SDS-PAGE electrophoreses. After that, the substances were transferred to a PVDF membrane. Rabbit anti-human Girdin polyclonal primary antibody (SC-133371, Santa Cruz Biotechnology) was added and kept in a shaker at 4°C overnight, and goat anti-rabbit secondary antibody (111-035-003, Jackson) was added for incubation at 37°C for 1 h. X-ray film was exposed, developed and fixed. The incubation and coloring of β-actin internal reference antibody were the same as the above mentioned methods, and the primary antibody and secondary antibodies used were mouse anti-human β-actin monoclonal antibody (BM0272, ABZOOM) and goat anti-mouse secondary antibody (111-035-008, Jackson), respectively.
5
Quantification and Visualization of PKP2 and KRT5 in Gingival Tissue
6
Whole-Cell Lysate Extraction and Western Blot
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Whole cell lysates were prepared using RIPA buffer (150mM NaCl, 1% w/v NP40, 50mM Tris pH 8.0, 0.5% w/v Sodium deoxycholate, 0.1% w/v SDS) 48 hours post-transfection and used for western blot analysis following published protocol [17 (link)]. Primary antibodies used are shown in S1 Table . The secondary antibodies used were goat anti-rabbit or a goat anti-mouse secondary antibody (Jackson Immunoresearch). The reported western blot results are representative of n = 3.
7
Sesamin Regulates Osteoblastic Differentiation
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After BMSCs osteoblastic differentiation, 1×105 cells/well was seeded in a 6-well plate for 24 hours, and then treated with sesamin (1 μM or 10 μM) for 3 days. We then collected the cells and extracted proteins using Pierce™ Universal Nuclease for Cell Lysis (Thermo) at 4°C for 20 minutes. Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 10% separating gel and 5% stocking gel, and then transferred to polyvinylidene difluoride (PVDF) membrane (Merch Millipore Corporation. Shanghai, China), which was blocked with 5% non-fat milk at 25°C for 2 hours; then incubated with mouse anti-RUNX2 antibody (1: 5000; ab76956, Abcam), mouse anti-osteocalcin (OCN) antibody (1: 3000; ab13420, Abcam), mouse anti-β-actin antibody (1: 5000; ab8226, Abcam), rabbit anti-low density lipoprotein receptor-related protein 5 (LRP5) antibody (1: 1000; ab38311, Abcam), mouse anti-glycogen synthase kinase-3β (GSK-3β) antibody (1: 2000; ab93926, Abcam), rabbit anti-β-catenin antibody (1: 5000; ab32572, Abcam), respectively, at 4°C, overnight. Goat anti-rabbit or goat anti-mouse secondary antibody (1: 3000; Jackson Immuno Research Laboratories, Inc., USA) was added and incubated at 25°C for 1 hour. Electrochemiluminescence (ECL; Millipore) was used for analysis.
8
Immunolabeling of Extracellular Vesicles for TEM Analysis
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Formvar-coated nickel grids with adherent EVs (see the previous section) were transferred for 10 minutes on single drops of 1% PBS-BSA (blocking buffer) to block unspecific sites. The grids were then incubated overnight at room temperature with mouse monoclonal anti-CD90 antibody (VMRD Inc., WA, USA) diluted 1 : 20 and rabbit anti-flotillin1 polyclonal antibody (Bioss Antibodies Inc., MA, USA) diluted 1 : 100 in blocking buffer. After several washes in PBS to remove the excess of antibody, the grids were incubated for 1 hour at room temperature, respectively, with goat anti-mouse secondary antibody and goat anti-rabbit, gold-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, PA, USA) diluted 1 : 40 in PBS with 1% BSA. Grids were finally washed in PBS, counterstained with 2% aqueous uranyl acetate for 5 min, washed with distilled water, and examined with a Philips EM 208 transmission electron microscope equipped with a digital camera (University Centre for Electron Microscopy (CUME) Perugia).
9
Immunoblotting Analysis of NEDD9 Protein
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Total protein was extracted in lysis buffer with protease inhibitors. After proteins were separated on a 10% SDS-PAGE gel, they were transferred to a cellulose acetate membrane. After blocking with 5% skim milk, the membrane was incubated with NEDD9 (1:1000, ab18056, Abcam, USA) and β-actin (1:10000, A5316, Sigma, USA) antibodies at 4 °C overnight. The membrane was subsequently washed and incubated with a goat anti-mouse secondary antibody (1:10,000, 115–035-003, Jackson, USA) for 2 h at room temperature. After the membrane was fully washed, chemiluminescence was performed for the development and visualization of the protein bands.
10
Purification and Enzymatic Assay of PHD2
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Flag-tagged PHD2 was expressed in insect cells and affinity-purified using anti-Flag, and the His-tagged HIF-2α oxygen-dependent degradation domain (ODDD) protein was expressed in E. coli and affinity-purified using NiNTA as described previously. 36, 33 A PHD2 enzymatic activity assay was performed to measure the radioactive CO 2 produced during the decarboxylation of 2-oxo[1-14 C]glutarate (Perkin-Elmer), which co-occurs with the substrate proline hydroxylation. 37 cycloheximide chase assay HEK cells were transfected with plasmids encoding pcD-NA3-HA-PHD2 (100-800 ng) in addition to pCMV-HA-empty vector for a total amount of 800 ng of transfected DNA. Twenty-four hours after transfection, cells were treated with cycloheximide (Sigma-Aldrich) at a final concentration of 100 μg/mL. Cells were harvested at different timepoints. An equal volume of protein lysates was analyzed by western blot assay using a mouse anti-HA antibody, anti-actin (Sigma) and a goat anti-mouse secondary antibody (Jackson Immunoresearch).
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