Journal: PLoS ONE

Article Title: Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel K v1.5

doi: 10.1371/journal.pone.0026524

Figure Lengend Snippet: Top : FHL1A, FHL1B and FHL1C protein structures (resulting from alternative splicing of the wild-type FHL1 gene), including the LIM domain architecture and functional domains ( RBP-Jk , recombination signal-binding protein 1 for J-kappa; NLS , nuclear localization signals; NES , nuclear export sequence) are shown for the corresponding variants. Bottom : Protein structures resulting from alternative splicing of the two different mutant FHL1 genes. An arrow in the fourth LIM domain of mutated FHL1A at position 224 (termed FHL1A p.C224W ) indicates the position of the amino acid exchange. The dotted line suggests alterations in the LIM-like architecture (termed MUT1 here). FHL1A p.G168fs is identical to FHL1C protein (termed MUT2 here) (G = glycine, fs = frame shift).

Article Snippet: The membranes were blocked (30 min) with TBST (10 mM Tris-HCl [pH 7.4], 140 mM NaCl, 0.1% [v/v] Tween-20) containing 3% (w/v) non-fat dry milk followed by incubation overnight at 4°C with polyclonal rabbit anti-human FHL1 antibody (1∶1000, cross-reacting with the fourth LIM domain [coded for by exon 4–6] of FHL1A, AVIVA Systems Biology, San Diego, CA, USA) or polyclonal goat anti-human K v1.5 (1∶200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as primary antibodies (diluted in 3% (w/v) BSA).

Techniques: Alternative Splicing, Functional Assay, Binding Assay, Sequencing, Mutagenesis

Journal: PLoS ONE

Article Title: Four and a Half LIM Protein 1C (FHL1C): A Binding Partner for Voltage-Gated Potassium Channel K v1.5

doi: 10.1371/journal.pone.0026524

Figure Lengend Snippet: (A) RNA was isolated from myoblasts from controls (WT1, WT2) and XMPMA patients (MUT1, MUT2) and the corresponding K v1.5 and FHL1 regions were amplified by RT-PCR. Primers, spanning from exon 1 to 2 (termed FHL1) amplify the mRNA stretch coding for the common N-terminus of FHL1 in all three FHL1 isoforms (FHL1A, FHL1B and FHL1C), while primers termed FHL1A or FHL1C specifically amplify mRNAs encoding FHL1A or FHL1C, respectively. (P = positive control: human fetal brain marathon cDNA). To ensure equal gel loading, RT-PCR for human HPRT1 was performed. One representative experiment out of three is shown. See for the primer sequences used. (B) Protein lysates from myoblasts from controls (WT1, WT2) and XMPMA patients (MUT1, MUT2) were subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes and immunoreactive bands were detected with anti-K v1.5 or anti-FHL1A as primary antibodies. After stripping, the membranes were incubated with anti-β-actin antibody. (K v1.5 = 68 kDa; FHL1A = 32 kDa; β-actin = 45 kDa). One representative experiment out of three is shown.

Article Snippet: The membranes were blocked (30 min) with TBST (10 mM Tris-HCl [pH 7.4], 140 mM NaCl, 0.1% [v/v] Tween-20) containing 3% (w/v) non-fat dry milk followed by incubation overnight at 4°C with polyclonal rabbit anti-human FHL1 antibody (1∶1000, cross-reacting with the fourth LIM domain [coded for by exon 4–6] of FHL1A, AVIVA Systems Biology, San Diego, CA, USA) or polyclonal goat anti-human K v1.5 (1∶200, Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) as primary antibodies (diluted in 3% (w/v) BSA).

Techniques: Isolation, Amplification, Reverse Transcription Polymerase Chain Reaction, Positive Control, SDS Page, Stripping Membranes, Incubation