Cells were rinsed with cold PBS and harvested in lysis buffer [31] (link). Then, the extractions were obtained and then centrifuged at 14,000 rpm for 30 min. Twenty-five micrograms of protein was loaded per lane and separated by SDS-PAGE, then transferred to nitrocellulose membranes and blocked overnight in 5% skim milk. Then, the membrane was incubated with primary antibodies at 4 °C and subsequently incubated with a secondary antibody for 2 h at room temperature. The primary antibodies for the blots are as follows: p-CaMKII (1:1000, Cell Signaling Technology, Inc.), p-cofilin (1:1000, Abcam plc), CaMKII (1:1000, Cell Signaling Technology, Inc.), cofilin (1:1000, Cell Signaling Technology, Inc.), Beclin1 (1:1000, Cell Signaling Technology, Inc.), LC3II (1:1000, Cell Signaling Technology, Inc.), ATG5 (1:1000, 1:1000, Abcam plc), Yap (1:1000, Cell Signaling Technology, Inc.), F-actin (1:1000, 1:1000, Abcam plc), G-actin (1:1000, 1:1000, Abcam plc), p-JNK (1:1000, Cell Signaling Technology, Inc.), JNK (1:1000, Cell Signaling Technology, Inc.), Parkin (1:1000, Cell Signaling Technology, Inc.), p-Parkin (1:1000, Cell Signaling Technology, Inc.), Bnip3 (1:1000, Cell Signaling Technology, Inc.), p62 (1:1000, 1:1000, Abcam plc) and SERCA (1:1000, Cell Signaling Technology, Inc.)
G actin
Manufactured by Abcam
Sourced in United States
G-actin is a globular protein involved in the formation of actin filaments, which are essential structural components of the cytoskeleton. It is the monomeric form of actin and plays a crucial role in cellular processes such as cell motility, cell division, and muscle contraction.
Automatically generated - may contain errors
Lab products found in correlation
F actin, by Abcam (4 mentions)
P cofilin, by Abcam (2 mentions)
Gapdh, by Abcam (2 mentions)
Camkii, by Cell Signaling Technology (1 mentions)
Serca, by Cell Signaling Technology (1 mentions)
Beclin 1, by Cell Signaling Technology (1 mentions)
P jnk, by Cell Signaling Technology (1 mentions)
Cofilin, by Cell Signaling Technology (1 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Bnip3, by Cell Signaling Technology (1 mentions)
P camkii, by Cell Signaling Technology (1 mentions)
P parkin, by Cell Signaling Technology (1 mentions)
Parkin, by Cell Signaling Technology (1 mentions)
P enos, by Abcam (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor tablet, by Roche (1 mentions)
Cofilin, by Abcam (1 mentions)
P camkii, by Abcam (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Mmp13, by Abcam (1 mentions)
6 protocols using g actin
1
Western Blot Analysis of Cell Signaling
2
Protein Expression Analysis Protocol
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Cells were lysed with RIPA buffer (65 mM Tris HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS) with protease inhibitor (04693132001, Roche) and phosphatase inhibitor tablets (4906837001, Roche). Protein concentration was quantified with a BCA kit. Similar amounts of protein were separated by 8-12% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore), and incubated overnight at 4 °C with corresponding primary antibodies: F-actin (1:1000, Abcam, #ab130935), G-actin (1:1000, Abcam, #ab200046), p-CaMKII (1:1000, Abcam, #ab124880), cofilin (1:1000, Abcam, #ab54532), CaMKII (1:1000, Abcam, #ab52476), p-cofilin (1:1000, Abcam, #ab283500), ET-1 (1:1000, Abcam, #ab178454), eNOS (1:1000, Abcam, #ab199956), p-eNOS (1:1000, Abcam, #ab215717), SGLT2 (:1000, Abcam, #ab37296), Fak (1:1000, Abcam, #ab40794), Src (1:1000, Abcam, #ab133283), GAPDH (1:1000, Abcam, #ab8245), XO (1:1000, Abcam, #ab109235), SERCA2 (1:1000, Abcam, #ab150435), Fak (1:1000, Abcam, #ab40794), Src (1:1000, Abcam, #ab133283), Tom20 (1:1000, Abcam, #ab186735), Drp1 (1:1000, Abcam, #ab184247), and SERCA2 C674-SO3H (#A300-BL2103; Bethyl Laboratories, Inc., Montgomery, TX, USA). Suitable secondary antibodies were next applied for 1 h at room temperature. Signals were detected on a ChemiDoc imaging system (Bio-Rad, Hercules, CA, USA).
3
Arteriogenesis Imaging of Coronary Micrangium
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Immunofluorescence confocal microscopy was used to observe the arteriogenesis of the coronary micrangium. Tissue sections were incubated overnight at 4 °C with first primary monoclonal antibodies against G-actin (1:100, rabbit polyclonal) and α-SMA (1:100, rabbit polyclonal, both from Abcam Inc., Cambridge, Massachusetts, USA). The fluorescein isothiocyanate (FITC)-labeled antibody (1:100, KPL, USA) and CyTM3-labeled antibody (1:100, KPL, USA) were incubated for 30 min at room temperature. Nuclei were stained with DAPI (Boster Biotechnology Co. Ltd, Wuhan, China). Controls were performed with/without the primary antibody or with/without the secondary antibody on the three group tissues, and were included in all experiments to correct for background fluorescence. For fluorescence analyses, an Olympus FV1000 confocal microscope (Olympus Tokyo, Japan) was used43 (link).
4
Protein Expression Analysis by Western Blot
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The cells were lysed, and the protein samples were collected. Next, the protein concentration was quantified using the bicinchoninic acid method. The denatured proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h, followed by incubation with the following primary antibodies overnight at 4°C: ACAN (1:100, 1 μg/mL, Abcam, USA), COL2A1 (1:5000, 1 μg/mL, Abcam, USA), SOX9 (1:5000, 0.5 μg/mL, Abcam, Britain), MMP13 (1:3000, 0.5 μg/mL, Abcam, Britain), F‐actin (1:2000, 0.5 μg/mL, Abcam, USA), G‐actin (1:1000, 0.5 μg/mL, Abcam, USA), GAPDH (1:5000, 0.5 μg/mL, Abcam, USA), LATS1 (1:5000, 1 μg/mL, Abcam, USA), p‐LATS1 (Tyr1079, 1:1000, 1 μg/mL, Cell Signaling Technology, USA), YAP (1:1000, 0.5 μg/mL, Cell Signaling Technology, USA), p‐YAP (Ser127, 1:1000, 1 μg/mL, Cell Signaling Technology, USA) and α‐catenin (1:500, 1 μg/mL, BD Transduction Laboratories, USA). Next, the membrane was incubated with the secondary antibody (1:5000, Cell Signaling Technology, USA) for 1 h at room temperature. Thereafter, the membrane was washed three times with TBST, and then the proteins were detected using an imaging system.
5
Immunohistochemical Analysis of Muscle Tissue
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Frozen tissue sections were fixed with 10% formalin, and cultured cells were fixed with 4% paraformaldehyde. All of the primary antibodies—lamin A/C (Santa Cruz), Pax7 (DHSB), CD68 (Abcam), PDGFR‐α (Abcam), fast‐type myosin heavy chain (f‐MHC) (Abcam), Sun1 (Novus Biologicals), Sun2 (Abcam), p21 (Cell signaling), γ‐H2AX (Cell Signaling), RhoA (Santa Cruz and Abcam), H3K9me3 (Abcam), and H3K27me3 (Abcam)—were used at a 1:100 to 1:300 dilution. All slides were analyzed via fluorescence microscopy (Nikon Instruments Inc.) and photographed at 4–40 × magnification. F‐actin was stained with Alexa Fluor 488 Phalloidin or Alexa Fluor 594 Phalloidin (Thermo Fisher). G‐actin was detected with an antibody to DNase I which specifically bind to G‐actin (Abcam). The cell nuclei were stained with DAPI. Fibrosis formation in muscle tissues was visualized by Masson trichrome staining with the Trichrome Stain (Masson) Kit (Sigma‐Aldrich). Sections were incubated in Weigert's iron hematoxylin working solution for 10 min and rinsed under running water for 10 min. Slides were transferred to Biebrich scarlet‐acid fuchsin solution for 15 min before incubation in aniline blue solution for another 5 min. Slides were then rinsed, dehydrated, and mounted as earlier. The ratio of the area of fibrotic collagen (blue) to the area of normal muscle (red) was quantified to measure fibrosis formation.
6
Immunoblotting Analysis of Cytoskeletal Proteins
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The cell lysate was collected and 20 μg of protein was subjected to SDS–PAGE, and then proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, United States). TBS/T (0.1%) containing 5% non–fat milk was used to block non–specific binding, the membrane was incubated in different primary antibodies: CFL1 (CST #5175), F–actin (Abcam#ab130935), G–actin (Abcam#ab123034), GAPDH (Abcam#ab128915). All antibodies were used at a 1:1,000 dilution, except for GAPDH, which was used at a 1:10,000 dilution. The membrane was washed with TBS/T four times to remove the unbound antibody and then incubated with the HRP–conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized with an ECL kit (Pierce, Rockford, IL, United States).
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