Cultured and pre-treated PMϕ (n ≥ 2 ∗ 106 cells per treatment group) were fixed and permeabilized with pre-chilled methanol–acetone (ratio 1:1). Non-specific antibody binds were blocked with 1% BSA in PBS for 1 h. PMϕ were then incubated with GRK5 primary antibody (ab64943; Abcam) at 4°C overnight, washed with PBS, and probed with R-PE-conjugated secondary antibody (SA00008-2; Proteintech) at room temperature for 1 h. Next, cells were washed with PBS, conditioned with 0.5% BSA in Hanks’ balanced salt solution, and the cytoplasmic membranes were stained with cholera toxin B (CTxB) (C34775, Thermo Fisher Scientific) for 30 min at 4°C. DAPI nuclei staining were done and followed by imaging and assessment of the ratios of nucleic and cytoplasmic GRK5 expressions with ImageJ (n = 12–15 cells per four mice).
Ab64943
Manufactured by Abcam
Ab64943 is a recombinant rabbit monoclonal antibody that recognizes human HSP90 alpha. This antibody is suitable for use in Western blotting, immunohistochemistry, and immunoprecipitation applications.
Automatically generated - may contain errors
Lab products found in correlation
R pe conjugated secondary antibody, by Proteintech (1 mentions)
Zoe fluorescent cell imager microscope, by Bio-Rad (1 mentions)
Dapi fluorescent dye, by Merck Group (1 mentions)
Ab5694, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Ab205718, by Abcam (1 mentions)
Chemiluminescence assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab8227, by Abcam (1 mentions)
Ab3442, by Abcam (1 mentions)
Ab84593, by Abcam (1 mentions)
Ab64644, by Abcam (1 mentions)
Ab25916, by Abcam (1 mentions)
Ab182136, by Abcam (1 mentions)
Collagen type 1, by Proteintech (1 mentions)
P nf κb, by Cell Signaling Technology (1 mentions)
Anp sc 515701, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Gapdh, by Proteintech (1 mentions)
5 protocols using ab64943
1
GRK5 Expression in Macrophages
2
Immunofluorescence Imaging of Fibroblasts and Cardiomyocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
3T3 fibroblasts, either WT or stable expressing GRK5-K215R mutation and H9c2 cells, overexpressing GRK5-WT, GRK5-ΔNLS, and GRK5-ΔNES, were fixed in 4% PFA for 10 minutes and then washed 3 times in ice cold phosphate-buffered saline and permeabilized with 0.2% Triton X-100. Then, 3T3 and H9c2 were incubated with 1% BSA for 30 min and incubated overnight at 4 °C with an anti-αSMA (ab5694, abcam; 1:200) or with an anti-GRK5 antibody (ab64943, Abcam, 1:200), respectively. Next, cells were incubated with the respective secondary antibodies (1:200). After staining, the sections were incubated with the DAPI fluorescent dye (Sigma-Aldrich). Images were acquired using ZOE Fluorescent Cell Imager Microscope (Bio-Rad Laboratories).
3
Quantification of GRK5 Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The present assay was used to detect the protein level of GRK5, with β‐actin as loading control. 16HBE, H1975, or H2342 cells were lysed in RIPA buffer (Beyotime) to extract protein samples, followed by quantification utilizing a bicinchoninic acid assay (BCA) kit. Then, 40‐μg samples were subjected to separation through sodium dodecyl sulfate (SDS)‐polyacrylamide gels (PAGE) and transfer onto polyvinylidene difluoride membranes (Thermo Fisher Scientific). The membranes were then blocked in skim milk, incubated with primary antibody against GRK5 (ab64943; Abcam) or β‐actin (ab8227; Abcam) then incubated with secondary antibody (ab205718; Abcam). Finally, protein signals were activated by feat of a chemiluminescence assay kit (Thermo Fisher Scientific) and analyzed exploiting Quantity One software.
4
Protein Expression Analysis in Cardiac Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apical myocardia harvested from four to six hearts per treatment group were homogenized in a lysis buffer containing phosphatase and proteinase cocktail inhibitor, in a ratio of 100:1:1, respectively. Lysates of normalized concentrations treated with reducing agents were denatured at 100°C for 10 min and separated on SDS-PAGE gels. The transferred protein bands were blocked and immunoblotted overnight in the following primary antibodies: β1AR (ab3442; Abcam), β2AR (ab182136; Abcam), MEF2 (ab64644; Abcam), GRK5 (ab64943; Abcam) GATA4 (ab84593; Abcam), NFAT (ab25916; Abcam), AC5 (PAC-501AP; FabGennix), AC6 (PAC-601AP; FabGennix), AC7 (PAC-701AP; FabGennix), ANP (sc-515701; Santa Cruz Biotechnology), BNP (sc-271185; Santa Cruz Biotechnology), GRK2 (sc-13143; Santa Cruz Biotechnology), pNF-κB (3033T; Cell Signaling Technology), NF-κB (8242T; Cell Signaling Technology), Cleaved Caspase-3 (9661T; Cell Signaling Technology), Collagen Type I (14695-1-AP, Proteintech), Collagen Type III (13548-1-AP, Proteintech), and GAPDH (10494-1-AP; Proteintech). Immunoblots were performed in triplicates and normalized with their respective loading controls.
5
Quantifying GRK5 Localization in PM Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fixation and permeabilization of pre-treated PMΦ (n > 1*106 cells per group) were done using pre-chilled methanol-acetone (ratio 1:1). The cells were blocked with 1% BSA, incubated overnight at 4°C with GRK5 antibody (ab64943; Abcam), and probed with R-PE-conjugated antibody (Proteintech; SA00008-2) at room temperature for 1 h. Next, the cells were wash and conditioned with 0.5% BSA in Hanks' balanced salt solution. Cholera toxin B (CTxB) (Thermo Fisher. C34775) was used to stain the cytoplasmic membranes for 30 min at 4°C and then counterstained with DAPI. GRK5 localization and expression ratios were ascertained using ImageJ (n = 12–15 cells per 4 mice).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!