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. 2014 Aug 13;34(33):11048–11066. doi: 10.1523/JNEUROSCI.3986-13.2014

Figure 6.

Figure 6.

Effect of hnRNPH1 siRNA on exon 2 inclusion/skipping. A, Effect of hnRNPH1 siRNA on the expression of hnRNPH1 protein in Be(2)C cells. An siRNA against hnRNPH1 (si-H1) and control siRNA (si-Ctr; Ambion, Life Technologies) were transfected into Be(2)C cells using NeuroMag reagent as described in Materials and Methods. Lanes 1 and 3: 10 nm siRNA; lanes 2 and 4: 25 nm siRNA. After 48 h of transfection, proteins were extracted from whole cells and used for Western blot analysis with hnRNPH1 or actin antibody, as described in Materials and Methods. B, Quantification of Western blots. Images on the films were analyzed on the ChemiDoc Image System. Band intensities of hnRNPH1 were obtained by adjusting with those of actin, and normalized with that of 10 nm si-Ctr (lane 1). All bars are the mean ± SEM. One-way ANOVA was used (n = 3 each). Red star: compared with lane 1; green star: compared with lane 2; blue star: compared with lane 3. *p < 0.05; ***p < 0.001. C, Effect of hnRNPH1 siRNA on exon 2 inclusion/skipping by regular qRT-PCR. RNA extracted from Be(2)C cells transfected with 25 nm of si-Ctr or si-H1 alone (Endogenous), or combined with BAC minigene construct G31 (hBAC-G31) was used in regular RT-PCR with SP-1 and AP-1 primers for the variants and G3PDH primers. Bands for hMOR-1, hMOR-1Z, and hMOR-1S are shown by boxes labeled as 1/2/3/4, 1/3/4, and 1/4, respectively. D, Effect of hnRNPH1 siRNA on exon 2 inclusion/skipping by qRT-PCR. The same cDNAs in C were used in SYBR green qPCR, as described in Materials and Methods. hE1–2 mainly represents hMOR-1. The fold change was calculated through 2-ΔΔC(t) format, where the ΔΔC(t) value was the difference of ΔC(t) values between the si-H1 and the si-Ctr samples, so the expression level in the si-Ctr samples is always 1. Bars represent the mean ± SEM. Student's t test was used (for endogenous variants: n = 6 each for hE1–2, n = 4 each for hMOR-1S, and n = 3 each for hMOR-1Z; for exogenous variants: n = 6 each for hE1–2, n = 9 each for hMOR-1S, and n = 6 each for hMOR-1Z). *p < 0.05; **p < 0.01; ***p < 0.001.