Oligomer distribution of recombinant MBL-A is altered in LH3^−/− knockout MEF cells.

<p>(A) Recombinant rat MBL-A produced in LH3 manipulated MEFs was separated under non-reducing and non-heat-denaturing conditions on SDS-PAGE and immunoblotted. Different oligomeric pattern was seen in MBL-A produced in LH3^−/− knockout MEFs compared with the other samples. The migration positions of covalent oligomeric forms of MBL-A are indicated on the right. Band marked with # represents four-chain covalent species of polypeptides. Representative samples are shown. (B) Quantification of MBL-A bands from immunoblots confirmed the change in the oligomer distribution of MBL-A produced in LH3^−/− knockout MEFs. Intensity of MBL-A band around molecular marker 150 kDa (marked with #) was quantified instead of the 2×3 band in LH3^−/− knockout MEFs. The values represent the average ± SD of six to eight experiments. (C) The oligomeric forms of recombinant rat MBL-A produced in LH3 manipulated MEFs were separated with a gel filtration chromatography and quantified from immunoblots. The gel filtration elution profile of MBL-A produced in LH3^−/− knockout MEFs also differed from wild type. Double transfection with MBL-A and LH3 constructs normalized the elution profile in LH3^−/− knockout MEFs. Equal volumes of concentrated cell culture media were used in all analysis. The average elution profiles of four to five experiments are shown. The elution positions of molecular weight markers are indicated. Abbreviations: WT = wild type; KO = LH3^−/− knockout; LH3 = full length LH3; LH3-N = amino-terminal fragment of LH3; MUT = LH mutant.