IMAGE

Fig. 3

ID
ZDB-IMAGE-180126-32
Source
Figures for Lee et al., 2017
Image
Figure Caption

Fig. 3

TMEM67 mutants show decrease in protein function. (a) HEK293T cells were transfected with plasmids individually encoding WT, p.Gly132Ala or p.Tyr920ThrfsX40 TMEM67, all fused C-terminally to a FLAG epitope, and processed for WB with anti-FLAG antibody. Anti-actin antibody was used for a loading control. Shown is a representative image of three independent experiments. Full-length blots are presented in Supplementary Fig. S1. (b) The transfected cells in (a) were processed for quantitative real-time PCR with TMEM67-specific primers. Expression levels of TMEM67 were normalized to those of ACTIN. ***P < 0.001 by the two-tailed Student’s t-test (n = 3). (c) The HEK293T cells transfected as described in (a) were treated with DMSO (dimethyl sulfoxide; vehicle control) or MG132 (25 μM) for 4 hr before harvest and processed for WB as described in (a). Cropped blots are presented. (d) A schema showing the binding site of tmem67 splice-blocking morpholino (MO [e2i2]). Dashed lines indicate splicing events. Not drawn to scale. E: exon; FP: forward primer; RP: reverse primer. (e) One-cell stage zebrafish embryos were injected with tmem67 MOs and processed for RT-PCR. β-actin was used as a loading control. Arrow indicates a PCR product with deletion of a part of exon2. (f) An electropherogram of an exon junction area of the PCR product indicated by arrow in (e). (g) One-cell stage zebrafish embryos were injected with either control MOs or tmem67 MOs [e2i2] and imaged at 2.5 days post-fertilization (dpf). For a rescue experiment, one-cell stage zebrafish embryos were sequentially injected with tmem67 MO [e2i2] and RNA encoding the indicated TMEM67 mutant and imaged at 2.5 dpf. Dashed lines mark the hindbrain ventricles. Arrows indicate enlarged ventricles (hydrocephalus). p.Tyr920* represents p.Tyr920ThrfsX40. Scale bar = 100 μm. (h) Embryos with hydrocephalus were counted in each group in (g). The hindbrain ventricles larger than 5,000 μm2 were considered to be hydrocephalic. *P < 0.05; ***P < 0.001 by the two-tailed Student’s t-test; NS: not significant. Number of larvae used in the analysis of each group is over 40.

Figure Data
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Sci. Rep.