Mammalian Expression of Rat MBL-A and Human LH3

Mammalian expression vectors pcDNA3 (Invitrogen) encoding a untagged or a carboxy-terminally FLAG-tagged rat MBL-A [23] (link) and pcDNA3 containing the full length human LH3 or the amino-terminal part of LH3 to the amino acid 290 with a LH3 signal peptide and a c-Myc-tag at the amino-terminus [5] (link),[24] (link) were transfected into LH3^−/− knockout, LH mutant and wild type MEFs using a MEF 1 Nucleofector Kit and Amaxa Nucleofector technology (Lonza). The following day the culture medium was changed to serum free DMEM, penicillin/streptomycin, and 50 µg/ml ascorbic acid, and the cells were cultured for 48 h. The medium was collected and concentrated by Amicon Ultra centrifugal filters (10 kDa MWCO, Millipore).

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Risteli M., Ruotsalainen H., Bergmann U., Venkatraman Girija U., Wallis R, & Myllylä R. (2014). Lysyl Hydroxylase 3 Modifies Lysine Residues to Facilitate Oligomerization of Mannan-Binding Lectin. PLoS ONE, 9(11), e113498.

Publication 2014

pcDNA3 MEF 1 Nucleofector Kit Amaxa Nucleofector technology Amicon Ultra centrifugal filters

Amino acid Ascorbic acid Cells Human Mammalian Penicillin Serum Signal peptide Streptomycin Vectors

Corresponding Organization : University of Oulu

Other organizations : University of Leicester

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Variable analysis

independent variables

Transfection of LH3-/- knockout, LH mutant and wild type MEFs with plasmids encoding untagged or FLAG-tagged rat MBL-A, and plasmids encoding full length human LH3 or the amino-terminal part of LH3 to the amino acid 290 with a LH3 signal peptide and a c-Myc-tag at the amino-terminus

dependent variables

Medium collected and concentrated after 48 hours of culture

control variables

Cell type (LH3-/- knockout, LH mutant and wild type MEFs)
Culture medium changed to serum free DMEM, penicillin/streptomycin, and 50 µg/ml ascorbic acid
Cells cultured for 48 hours

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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