Whole-cell protein extracts were obtained with radio-immunoprecipitation assay buffer following standard protocols. The protein extracts were denatured and 40–50 μg of each sample were separated on 12% SDS–PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% non-fat milk (LabScientific, Inc.) in TBS-Tween buffer, the membranes were probed with antibodies against E-cadherin (1:250, Santa Cruz), N-cadherin (1:250, Santa Cruz), Smad7 (1:200, Santa Cruz), ACTR-IIA (1:500, Santa Cruz), ACTR-IIB (1:500, Santa Cruz), actin (1:1,000, Santa Cruz), keratin 8 (1:250, Epitomics), Col3A1 (1:200, Abcam), Smad2 (1:500, Cell Signaling), P-Smad2 (1:250, Cell Signaling), Smad3 (1:200, Cell Signaling), P-Smad3 (1:100, Cell Signaling) and fibronectin (1:1,000, BD Transduction Lab). Membranes were exposed using enhanced chemiluminescence (Roche) method following manufacturer’s instructions. Full scans of all western blots are included in Supplementary Fig. S10 .
Actr iib
Manufactured by Santa Cruz Biotechnology
ACTR-IIB is a recombinant protein that functions as an activin type-2B receptor. It is commonly used in cell-based and biochemical assays to study the signaling pathways involving the activin type-2B receptor.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by BD (1 mentions)
Col3a1, by Abcam (1 mentions)
Keratin 8, by Abcam (1 mentions)
N cadherin, by Santa Cruz Biotechnology (1 mentions)
Smad7, by Santa Cruz Biotechnology (1 mentions)
Smad2, by Cell Signaling Technology (1 mentions)
P smad2, by Cell Signaling Technology (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
2 protocols using actr iib
1
Western Blot Analysis of EMT Markers
2
Immunostaining and Microscopy Analysis of HK-2 Cells
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HK-2 grown on chamber slides to sub-confluence were exposed to different stimuli as above described. After five-minute fixation in cold methanol, cells were incubated with anti-MSTN or anti-Nox-4 (Santa Cruz Biotechnology) or anti-fibronectin (Merck) antibodies. Immunostaining was performed as described previously20 (link),21 (link). Slides were counterstained with haematoxylin and examined by light microscopy.
For immunofluorescence, HK-2 were incubated with ACTR IIB (Santa Cruz Biotechnology) antibody, subsequently with FITC antibody (Merck) and then observed under the fluorescence microscopy.
For immunofluorescence, HK-2 were incubated with ACTR IIB (Santa Cruz Biotechnology) antibody, subsequently with FITC antibody (Merck) and then observed under the fluorescence microscopy.
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