Fig 1.
The MIC2655kDa form is neither sensitive to knockdown nor knockout of MIC26.
A) Western blot (WB) analysis of HEK293 cells knocked down for MIC26 reveal the loss of mitochondrial MIC2622kDa but not MIC2655kDa. In order to downregulate MIC26, three siRNAs were used. B)–E) WB analysis of cell lysates and secreted proteins, using WT and MIC26 KO HepG2 cells, were done using four anti-MIC26 antibodies. All four WBs show the presence of intracellular mitochondrial MIC2622kDa form in WT cells but not in MIC26 KO cells as expected. However, unexpected and inconsistent results were obtained w.r.t the 55 kDa protein. (B) Antibody #1 detects an intra- and extracellular 55 kDa band in WT as well as MIC26 KOs. (C) Antibody #2 detects only an intracellular protein band around 70 kDa. (D) Antibody #3 identifies an intracellular and secreted protein (in culture medium) at ≈ 70 kDa in both WT and MIC26 KO HepG2 cells. The considerable increase in ≈ 70 kDa protein correlates to the production and secretion of albumin by human HepG2 cells, which is nonspecifically recognized by this antibody. (E) Antibody #4 is not recognizing a 55 kDa band in WT and MIC26 KO HepG2 cells.
Fig 2.
MIC26 KOs reveal the nonspecific nature of the 55 kDa band in various human cell lines.
WB analysis of WT, MIC26 and MIC27 KO cell lysates, obtained from HEK293, HepG2, HeLa and HAP1 cells are shown. MIC26 KOs reveal a loss of mitochondrial MIC2622kDa but not the 55 kDa band. There is no protein band detected in WT and MIC27 KOs at 55 kDa region when anti-MIC27 antibody was used. A) In MIC26 KOs of HEK293 cells, a loss of MIC2622kDa is observed contrary to an increased amount of 55 kDa protein in WT. B) The KO of MIC26 in HepG2 cells leads to the loss of the 22kDa form but unchanged expression of the 55 kDa band. C) HeLa cells show similar results to HEK293 cells w.r.t loss of the 22 kDa form and an increased expression of the 55 kDa band. D) In HAP1 cells, the MIC26 KO leads to a loss of the mitochondrial MIC2622kDa protein contrary to unaltered expression of the 55 kDa protein in WT cells.
Fig 3.
Exogenous expression of MIC26-GFP or MIC26-myc does not result in the formation of corresponding MIC2655kDa-GFP or MIC2655kDa-myc protein.
WB analysis of HEK293 cells expressing MIC26-GFP or MIC26-myc along with various theoretically predicted glycosylation defective mutants. A) Overexpression of mitoGFP (control), MIC26-GFP and the single mutants of MIC26-GFP namely MIC26S34A-GFP, MIC26S41A-GFP and MIC26S50A-GFP in HEK WT cells leads to detection of the endogenous MIC2622kDa protein, a MIC2622kDa-GFP protein (≈ 50 kDa protein below the 55 kDa protein marked in red asterisks) and the endogenous 55 kDa band when antibody #1 was used. B) Immunoblotting with anti-GFP antibody shows a ≈ 50 kDa band detecting MIC2622kDa-GFP. However, no band correlating to a MIC2655kDa-GFP protein (≈ 80 kDa) could be observed. C) Exogenous expression of MIC26-myc in HEK293 MIC26 KO leads to the formation of MIC2622kDa-myc protein with an expected slight shift in molecular weight compared to the expression of untagged MIC26. However, MIC2655kDa-myc protein was neither detected with anti-MIC26 nor anti-myc tag antibody. Expression of the glycosylation defective triple mutant, MIC26S34A/S41A/S50A-myc, where the respective serine amino acids were mutated to alanine (S34A, S41A, S50A), showed unaltered 55 kDa protein levels.
Fig 4.
Unspecific nature of the 55 kDa band is neither cell line- or species-specific.
WB analysis of human WT HEK293 and HeLa cell lysates along with murine liver tissue lysates and plasma comparing two different anti-MIC26 antibodies: A) Antibody #1 detects MIC2622kDa and an unspecific 55 kDa protein in HEK293 and HeLa as well as in murine liver. B) Antibody #2 exclusively detects the mitochondrial MIC2622kDa, but no 55 kDa band. C) Antibody #1 does not recognize a mitochondrial MIC2622kDa and 55 kDa band in plasma samples from two mice. D) Antibody #2, like antibody #1, does not recognize any protein in murine plasma samples. E) Antibody #1 detects the MIC2622kDa as well as an unspecific 55 kDa band in C2C12 cell line along with different murine tissue lysates but not plasma. F) Antibody #2 detects MIC2622kDa in mouse cell lines and tissues but not the 55 kDa band. C2C12-A represents C2C12 cells lysed with RIPA buffer and C2C12-B represents C2C12 cells which were mechanically lysed.
Fig 5.
The diabetic db/db mouse model does not show different levels of the exclusively mitochondrially localized MIC26 and MIC27 proteins in heart and liver.
WB analysis of lysates from heart (A) and liver (C) tissue, along with associated quantification showing protein levels of MIC26 and MIC27 (B and D) from control db/+ and diabetic db/db.BKS mice, reveal no significant differences in amounts of MIC26 and MIC27. E) Mic26 and Mic27 transcript levels are not changed in liver of control db/+ and db/db.BKS diabetic mice.