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. 2012 Jun 18;13(6):7532–7549. doi: 10.3390/ijms13067532

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© 2012 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.

This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).

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Effects of amino acid substitutions in Pocket 3 on the binding abilities to PLSCR3 by GST-ALG-2 pulldown assays. HEK293/ALG-2_KD cells were transfected with expression vectors for GFP-fused PLSCR3 of wild-type, ABS2-deletion mutant (ΔABS2), ABS1-deletion mutant (ΔABS1) and unfused GFP used as a negative control (Ctrl). Supernatants of cell lysates were subjected to pulldown assays using GST-fused ALG-2 proteins of wild-type (WT), alternatively spliced isoform lacking Gly¹²¹Phe¹²² (ΔGF122), mutant of Leu⁵² to alanine substitution (L52A) and mutant of Phe¹⁴⁸ to serine substitution (F148S) as well as unfused GST used as a negative control in the presence of 100 μM CaCl₂. Proteins bound to the glutathione Sepharose beads (pulldown fractions) were analyzed by Western blotting using an anti-GFP antibody. Protein samples corresponding to 1% of total cleared cell lysates (Cell Lysate Sup) and 20% of pulldown fractions (Pulldown) were loaded to each lane on 10% gels of SDS-PAGE.