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. 2014 May 23;289(28):19466–19476. doi: 10.1074/jbc.M113.530972

FIGURE 1.

FIGURE 1.

Screening of NLGN isoforms expression and validation of NLGN silencing and overexpression efficiency in ECs. A, NLGN1, -2, -3, -4X, and -4Y expression is presented for primary cells (human umbilical vein and human umbilical artery ECs) and organs (brain, heart, and carotid). NLGN mRNA levels were assessed by qRT-PCR and the graph shows the single threshold cycle (Ct), which is inversely correlated to the expression level. The data revealed NLGN1 and NLGN2 as the most expressed isoforms in the ECs. Values are mean ± S.E. of three independent experiments. B, immunoprecipitation assay was performed in ECs using an anti-pan-NLGNs (L067) antibody and immunoblotting was conducted with a monoclonal antibody (4F9) able to recognize NLGN1 and NLGN2 isoforms (top panel). The membrane was then stripped and incubated with a polyclonal antibody recognizing only NLGN2 (bottom panel). The 120- and 100-kDa bands detectable in ECs correspond to NLGN1 and NLGN2, respectively. The images shown are representative of 1 of 3 reproducible experiments. C, left panel: qRT-PCR of NLGN1 and NLGN2 expression level on ECs transfected with control siRNA (SCRL), with two independent specific siRNA sequences targeting NLGN1 (A5 and A6) or with one specific siRNA sequence targeting NLGN2 (B9). Fold-change is calculated with respect to cells transfected with siRNA SCRL and values are expressed as mean ± S.E. (n = 3 independent experiments). One-way ANOVA with Bonferroni test: ***, p < 0.001. Right panel: qRT-PCR of NLGN1 expression level on ECs infected with the retroviral vector pBABE EMPTY (EMPTY) or containing the human cDNA sequence of NLGN1 (NLGN1). Results are expressed as fold-change relative to pBABE EMPTY. Values are mean ± S.E. (n = 3 independent experiments). One-way ANOVA with Bonferroni test: ***, p < 0.001. D, immunoprecipitation assay performed using the pan-NLGNs antibody (L067) in ECs transfected with control siRNA (SCRL) or NLGN1 siRNAs (A5 and A6) (left panel) or infected with pBABE EMPTY (EMPTY) or pBABE NLGN1 (NLGN1) retroviral vector (right panel). Immunoblottings (IB) were carried out using the monoclonal antibody 4C12, which specifically recognizes NLGN1. Images shown are representative of 1 of 3 experiments.