For immunoblot analysis, PC cells (1×106 in 10 mL medium) were seeded in a 100 mm culture dish and treated with 5 or 10 nM Dtxl or Dtxl equivalent SPION-Dtxl for 48 hrs. Cells were lysed with 2X SDS lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA). The process of extraction, quantification of proteins, and the Western blotting method were followed as described previously [39 (link)]. Primary antibodies [β-actin (#4967), BAD (#9292), Bax (#2772), Bcl-xL (#2762), Bcl-2 (#2876), p53 (#9282), Cleaved PARP (#5625), PSMA (#12815), and MDR1 (#13342)] and secondary antibody [horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit secondary antibody] (Promega) were used in this study. The protein bands were detected and imaged with the Lumi-Light Detection Kit (Roche, Nutley, NJ) using a BioSpectrum® 500 Imaging System. The densitometric studies of the acquired blots were analyzed using GelQuant® Software (DNR Bio-Imaging Systems, Jerusalem, Israel).
Bcl2 associated x (bax)
Manufactured by Promega
Sourced in Spain
Bax is a laboratory equipment product designed for use in research applications. It serves as a core functional component for various experimental procedures. The detailed technical specifications and intended use cases are not included in this response to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Lumi light detection kit, by Roche (1 mentions)
Sds lysis buffer, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Promega (1 mentions)
Cleaved parp, by Promega (1 mentions)
β actin, by Promega (1 mentions)
Gelquant software, by DNR Bio-Imaging Systems (1 mentions)
α tubulin, by Merck Group (1 mentions)
Horseradish peroxidase, by Bio-Rad (1 mentions)
P jnk, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Santa Cruz Biotechnology (1 mentions)
P erk1 2, by Promega (1 mentions)
2 protocols using bcl2 associated x (bax)
1
Immunoblot Analysis of PC Cells
2
Protein Extraction and Immunoblotting
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Total protein extracts were obtained by adding the lysis buffer: 25 mM HEPES pH 7.5, 0.3 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 20 mM β-glicerophosphate, 0.1 mM Na3VO4, 0.1% triton X-100; in the presence of protease inhibitors78 (link). Twenty μg of protein per sample were loaded and resolved using 8%, 10% or 15% SDS-PAGE polyacrylamide gels, and then transferred onto PVDF membranes, followed by immunodetection using appropriate antibodies, purchased from Santa Cruz Technology: Mcl-1 (sc-819), Cell Signaling Technology: BIM, BID, BAX (#9942), p-p38 (1:2000, #4631), p-p53ser15 (#9284), p-ERK1/2 (1:2000, #9106), p-CHK1ser345 and p-CHK2ser19 (#9931), Promega Corporation-Spain: p-JNK (#V7932), or Sigma-Aldrich: α-tubulin (1:10000, #TP026). Unless indicated, all antibodies were diluted 1:1000. Secondary antibodies conjugated with horseradish peroxidase were purchased from BioRad, and chemiluminescence detection was performed using ECL (Santa Cruz Biotechnology).
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