MIA PaCa-2 and PANC-1 cells (2×105 cells per well in 6-well plate) were treated with 1, 5, 10, and 20 μM of αM or γM. Cells were lysed in 200 μl RIPA Buffer (Thermo Scientific, Waltham, MA, USA) containing phosphatase inhibitor and protease inhibitor (Roche) for 20 min on ice following the manufacturer’s protocol. Protein concentrations were measured using BCA Protein Assay Kit (Thermo Scientific). Total protein (30 μg) was separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked with 5% of nonfat dry milk in TBST at room temperature for more than 1 h and incubated at 4°C overnight with the following primary antibodies: anti-human-Bax, PAPR, cleaved PARP, caspase-3, cleaved caspase-3, LC3II, SQSTM1/P62, AMPKα, phospho-AMPKα, mTOR, phospho-mTOR, p38 MAPK, and phospho-p38 MAPK from Cell Signaling Technology (Beverly, MA, USA), β-Actin from Bioworld Technology (St. Louis Park, MN, USA). Blots were then probed with HRP-conjugated anti-rabbit antibodies from Thermo Scientific or anti-mouse antibodies from Bethyl Laboratories (Montgomery, TX, USA). Blots were visualized using ECL solution (Thermo Scientific).
Anti human bax
Manufactured by Cell Signaling Technology
Sourced in United States, Italy
Anti-human-Bax is a primary antibody that specifically recognizes the Bax protein in human samples. Bax is a pro-apoptotic member of the Bcl-2 family and plays a key role in the intrinsic apoptosis pathway.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Phospho ampkα, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Hrp conjugated anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Ampkα, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Ecl solution, by Thermo Fisher Scientific (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Plan neofluar, by Zeiss (1 mentions)
Anti human lc3a b, by Cell Signaling Technology (1 mentions)
Anti human sod1, by Abcam (1 mentions)
Lsm 800 confocal system, by Zeiss (1 mentions)
Anti human caspase 1, by Abcam (1 mentions)
2 protocols using anti human bax
1
Apoptosis and Autophagy Regulation in Pancreatic Cancer
2
Immunofluorescence Analysis of hPDLSCs
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The hPDLSCs-CTR and hPDLSCs-MOR were processed as previously reported by Trubiani et al. (2012) [88 (link)]. Primary monoclonal antibodies anti-human Caspase 1 (1:200, rabbit) (Abcam, Milan, Italy), anti-human SOD1 (1:200, rabbit) (Abcam), anti-human Bax (1:100, rabbit) (Cell Signaling Technology, Milan, Italy), anti-human Bcl2 (1:200, rabbit) (Cell Signaling Technollogy, Milan, Italy), and anti-human LC3A/B (1:100, rabbit) (Cell Signaling Technology) were used, followed by Alexa Fluor 568 conjugated goat anti rabbit as secondary antibodies (1:200; ThermoFisher, Life Tech., Monza, MB, Italy) for 1 hr at 37 °C. Subsequently cells were incubated with AlexaFluor 488 phalloidin green fluorescence conjugate (1:200; ThermoFisher, Life Tech.), to evidence cytoskeleton actin. Cell nuclei were stained with TOPRO (1:200; ThermoFisher) for 1 hr at 37 °C. Glass coverslips were placed face down on glass slides and mounted with Prolong antifade (ThermoFisher, Life Tech.) [91 (link)]. Samples were observed by means of a Zeiss LSM800 confocal system, connected to an inverted Zeiss Axiovert 200 microscope equipped with a Plan Neofluar oil-immersion objective. Images were collected using an argon laser beam with excitation lines at 488 nm and a helium-neon source at 543 and 633 nm. Post-acquisition image analyses were carried out with a Zeiss ZEN software.
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