Panc-1 cells (5×106) were plated onto 15-cm dishes and attached overnight. The cells were treated with DMSO or 40 µM wogonin for 12 h at 37°C then lysed for 30 min in hypotonic lysis buffer (Beyotime Institute of Biotechnology) on ice and centrifuged (1,000 × g, 4°C, 15 min) as a whole-cell lysate. Whole-cell lysates were precleared with protein A-agarose (50% in PBS), and then incubated with antibodies against Beclin-1 or PI3K (#sc-11427 and #sc-134766; Santa Cruz Biotechnology, Inc.; dilution, 1:300) for 1 h at 4°C. The immunoprecipitates were captured on a protein-A agarose gel and then detected by immnuoblotting; whole-cell lysates and immunoprecipitates were separated by 12% SDS-PAGE and transferred to PVDF membranes for detection, as described above (17 (link)).
Hypotonic lysis buffer
Manufactured by Beyotime
Hypotonic lysis buffer is a solution designed to facilitate the lysis or breakdown of cells, particularly in preparation for protein extraction or other downstream applications. The core function of this buffer is to create an environment with a lower osmotic pressure compared to the interior of the cells, causing them to swell and rupture, releasing their contents.
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Lab products found in correlation
2 protocols using hypotonic lysis buffer
1
Wogonin Modulates Beclin-1 and PI3K in Panc-1 Cells
2
Western Blot Analysis of Podocyte Proteins
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For Western blot analysis, podocytes were lysed in hypotonic lysis buffer (Beyotime), and equal amounts of protein were denatured after heating at 95°C for 5 min and loaded on to an SDS/8% polyacrylamide gel. Separated proteins were subsequently transferred to a nitrocellulose membrane and blocked with 8% nonfat milk at room temperature for 1 h. Membranes with proteins were incubated with primary antibodies at 4°C overnight. The primary anti-MR antibody (1:100, Santa Cruz Biotechnology), anti-human integrin β1 polyclonal antibody (1:400, Bosider), anti-p85-PI3K antibody (1:800, Cell Signaling Technology), anti-p-Akt antibody (ser473, 1:800, Cell Signaling Technology), anti-t-Akt antibody (1:800, Cell Signaling Technology), anti-p-mTOR antibody (ser2448, 1:200, Santa Cruz Biotechnology), anti-Atg5 antibody (1:500, Santa Cruz Biotechnology) and β-actin antibody were used. The second antibodies (horseradish peroxidase conjugate goat anti-rabbit, 1:5000 in blocking solution) were added and incubated for 2 h at 4°C. All blots were developed using Western blotting detection system of enhanced chemiluminescence (Pierce Biotechnology).
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