Astrocytes were fixed with 4% paraformaldehyde at room temperature for 20 min, washed with PBS, and incubated with 10% normal goat serum and 0.1% Triton X-100 in PBS for 30 min. Next, cells were incubated overnight at 4°C with the primary antibody, washed with PBS, and then incubated with Alexa Fluor 488- or 568-conjugated secondary antibody (1:1000, Invitrogen) for 1 h at room temperature. Cells were then washed, mounted onto microscope slides in Vectashield mounting medium containing the nuclear dye, 4,6-diamidino-2-phenylindole (DAPI) (Vector laboratories, Burlingame, CA). The following primary antibodies were used: phospho-Smad2 #3108 and phospho-Smad3 #9520 (each 1:100, Cell Signaling); GFAP (1:400, Z0334, Dako); KCa3.1 (1:100, ab83740, Abcam); Ki67 (1:50, ab16667, Abcam).
Phospho smad3 9520
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-Smad3 #9520 is a rabbit monoclonal antibody that detects endogenous levels of Smad3 protein only when phosphorylated at Ser423 and Ser425.
Automatically generated - may contain errors
Lab products found in correlation
Ab16667, by Abcam (1 mentions)
Phospho smad2 3108, by Cell Signaling Technology (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Fibronectin 610077, by BD (1 mentions)
αsma a2547, by Merck Group (1 mentions)
Smad2 3 3102, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit g 21234, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide d8418, by Merck Group (1 mentions)
Tgf β 240 b, by R&D Systems (1 mentions)
β actin a5441, by Merck Group (1 mentions)
Sirt3, by Cell Signaling Technology (1 mentions)
Phospho smad2 3101, by Cell Signaling Technology (1 mentions)
Las 3000, by Fujifilm (1 mentions)
3 protocols using phospho smad3 9520
1
Immunofluorescence Staining of Astrocytes
2
Fibrosis-related Protein Detection Assay
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PAI-1 (614024) and fibronectin (610077) antibodies were obtained from BD Biosciences (Franklin Lakes, NJ, USA). Phospho-Smad3 (#9520) and Smad2/3 (#3102) antibodies were provided by Cell Signaling (Danvers, MA, USA). CTGF antibodies (SC-101586) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and TGF-β1 antibody (NBP1-80289) was obtained from Novus Biologicals (Littleton, CO). Horseradish peroxidase-conjugated goat anti-rabbit (G21234) and anti-mouse (G21040) antibodies were purchased from Invitrogen (Carlsbad, CA, USA). α-SMA (A2547) and β-actin (A5441) antibodies, MTT (M2128), and dimethylsulfoxide (D8418) were acquired from Sigma Chemicals (St. Louis, MO, USA). TGF-β (240-B) was purchased from R&D Systems (Minneapolis, MN, USA). CTGF antibody (sc-101586) obtained from Santa Cruz Biotechnology (Santa Cruz, CA). CCl4 (33650-0330) was received from Junsei Chemical (Kyoto, CO).
3
Western Blot Analysis of Fibrosis Markers
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Western blot analysis was performed as described previously [23 (link)]. Primary antibodies to α-SMA (A2547; mouse; dilution 1:1,000; Sigma Chemical Co., St Louis, MO, USA), ICAM-1 (sc-1511; goat; 1:1000) and fibronectin (sc-6953; goat; 1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), type I collagen (1310-01; goat; 1:1000; Southern Biotech, Birmingham, AL, USA), SIRT3 (5490S; rabbit), DRP1 (5391; rabbit), OPA1 (80,471; rabbit), phospho-Smad2 (3101; rabbit) and phospho-Smad3 (9520; rabbit) (1:1000; Cell Signaling Technology, Danvers, MA, USA), and Smad2/3 (07-408; rabbit; 1:1000; EMD Millipore, Billerica, MA, USA) were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; AP0063; rabbit; 1:2000; Bioworld Technology, Inc., St. Louis Park, MA, USA) was used as an internal control. All signals were analyzed by densitometric scanning (LAS-3000, Fuji Film, Tokyo, Japan).
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