Studies on Human Porin. VI. Production and Characterization of Eight Monoclonal Mouse Antibodies against the Human VDAC “Porin 31HL” and Their Application for Histotopological Studies in Human Skeletal Muscle

Abstract

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL.In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC 'Torin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin.Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines.

The expression level of the voltage-dependent anion channel controls life and death of the cell

“…Cell growth rates were monitored by using trypan-blue staining. The expression levels of mVDAC1 were followed by Western blot analysis of cell extracts using monoclonal anti-VDAC1 antibodies (21,22) and quantified by densitometry. As a control for protein amount in all samples, blotting with anti-actin antibodies was performed.…”

“…1C). Because the hVDAC1-shRNA used is specific for VDAC1, and specific anti-VDAC1 antibodies were used (21,22), the results suggest that VDAC1 is required for normal cell growth.…”

Porin Is Present in the Plasma Membrane Where It Is Concentrated in Caveolae and Caveolae-related Domains

“…5A). The purified protein was identified as porin by positive staining in Western blots with polyclonal Abs versus purified bovine heart mitochondrial porin or with monoclonal Abs versus a synthetic peptide reproducing the N terminus of HV-DAC1 (22,23). This result demonstrates the purification of porin molecules labeled by NHS-SS-biotin, coming from the plasma membrane of intact CEM cells.…”

“…The anti-porin antibodies were raised either against the whole purified porin from bovine heart mitochondria or against a synthetic peptide mimicking the N-terminal end of HVDAC1 (22). These antibodies did not show any cross-reactivity with sequences from HVDAC2 (23) nor with any cellular protein other than HVDAC1.…”

VDAC/porin is present in sarcoplasmic reticulum from skeletal muscle

“…2. Monoclonal antibodies, raised against the N-terminal part of the human B-lymphocytes VDAC [29], cross-react with two SR proteins of 35 and 30 kDa. We detected the two immunoreactive polypeptides of 35 and 30 kDa in SR in a ratio of about 10:1, while a much higher ratio is found in mitochondrial membranes (Fig.…”