Figure 3.

RMND1 Is Partially Reduced in VI-3 Fibroblasts

(A) qRT-PCR was performed for characterizing the expression level of RMND1 mRNA in VI-3 fibroblasts with the use of TaqMan Assays for RMND1 and ACTB (β-actin) transcripts (Applied Biosystems). Values are expressed as percentages of controls. Data are represented as the mean ± standard deviation and are the results of at least three different experiments. The asterisk (^∗) indicates a Student’s t test p < 0.05.

(B and C) Immunoblot for measuring the level of RMND1 in VI-3 fibroblasts. Proteins were extracted, and concentrations were measured with the BCA Protein Assay Kit (Pierce). Five micrograms of protein was electrophoresed in a SDS-12%-PAGE gel, transferred to Immun-Blot PVDF membranes (Biorad, Hercules, CA, USA), and probed with rabbit polyclonal RMND1 antibody (product #HPA031399, Sigma-Aldrich, St. Louis, MO) at a 1:1,000 dilution. A mouse monoclonal actin antibody (Sigma-Aldrich) was used at a 1:10,000 dilution. Protein-antibody interaction was detected with peroxidase-conjugated goat anti-mouse IgG antibody (1:5,000) (Sigma-Aldrich) with SuperSignal chemiluminiscence detection kit (Thermo Fisher Scientific, Waltham, MA). Quantitation of the bands was performed by densitometric analysis with the National Institutes of Health ImageJ software package (version 1.45). Values are expressed as percentages of controls. Data are represented as the mean ± standard deviation and are the results of at least three different experiments. The asterisks (^∗) indicate a Student’s t test p < 0.05. The following abbreviations are used: C1–C4, controls; and actin, β-actin.