Journal: Cerebral Cortex (New York, NY)

Article Title: Suppression of Cofilin function in the somatosensory cortex alters social contact behavior in the BTBR mouse inbred line

doi: 10.1093/cercor/bhae136

Figure Lengend Snippet: Viral suppression of Cofilin in the somatosensory cortex of BTBR mice. A) Mice were injected with pAAV5-CaMKIIα0.4-eGFP or pAAV5-CaMKIIα0.4-CofilinS3D-HA into the somatosensory cortex to drive expression of either eGFP, as a control, or the mutant inactive form of Cofilin (Cofilin S3D ) in excitatory neurons. An HA-tag was included to discriminate between mutant and endogenous Cofilin. B) Western blot analysis of virally delivered Cofilin S3D protein levels in the somatosensory cortex. An HA-tag antibody was used to detect the mutant inactive form of Cofilin ( n = 2). C, D) Injection site of the AAVs into the somatosensory cortex ( n = 8 for CofilinS3D and n = 7 for eGFP). Oval shaped areas in the left hemisphere (C + D) represent the overlapping virus injection sites in the somatosensory cortex for the tested animals. These areas represent the overlapping virus injection sites for the tested animals. Right hemisphere shows a representative staining of the virus in the somatosensory cortex. In general, autofluorescence of the eGFP proteins gives a stronger signal throughout the cortex, while antibody-based fluorescence of the HA-tag shows less staining in the deeper layers of the cortex.

Article Snippet: Mice were either injected with this AAV5.CaMKII0.4.mCofilin.S3D-HA.rBG or a control virus under the same promoter, a pAAV5-CaMKIIa-EGFP (eGFP) vector (Addgene, USA) (stock titer: 4.30 × 10 12 genome copies/mL).

Techniques: Injection, Expressing, Mutagenesis, Western Blot, Virus, Staining, Fluorescence

Journal: ASN neuro

Article Title: Caspase-mediated apoptosis in the cochleae contributes to the early onset of hearing loss in A/J mice.

doi: 10.1177/1759091415573985

Figure Lengend Snippet: Figure 5. Expression of caspase-3 in HEI-OC1 cells. (a) 2% agarose gel electrophoresis of the RT-PCR products of Gapdh, Cs, and Cdh23 in HEI-OC1 cells. Lane M, 50 bp DNA ladders; lanes 1–2, 3–4, and 5–6 represent RT-PCR products of Gapdh (100 bp), Cdh23 (228 bp), and Cs (144 bp), respectively. (b) Transcription levels of Cs and Cdh23 in HEI-OC1 cells transfected with shRNA. mRNA levels of Cs was significantly suppressed (p <.05) by Cs shRNA (Cs knockdown group) compared with that by control shRNA (control, limited to1 unit for the level of each gene). mRNA levels of Cs and Cdh23 in cotransfencted group (Cdh23 shRNA þCs shRNA) were also lower than those of the controls. (c) Caspase-3 levels determined by Western blotting. Caspase-3 expression levels were markedly elevated in Cs knockdown group or cotransfected group compared with those of controls, whereas were not elevated in Cdh23 knockdown group. (d) The average gray intensity of caspase-3 protein detected by Western blotting was standardized to that of b-actin. Caspase-3 expression levels in Cs knockdown group or cotransfected group were significantly higher than those of the controls. Data are shown as mean SD of triplicate experiments. *p <.05. shRNA ¼ short hairpin RNA; RT-PCR ¼ reverse transcription polymerase chain reaction; mRNA ¼ messenger RNA; Gapdh ¼ glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: When the cells reached 60% confluency in the culture dishes, the cells were transfected with Cs shRNA (Cs shRNA Plasmid, sc-96228-SH, Santa Cruz Biotechnology), Cdh23 shRNA (cadherin 23 shRNA Plasmid, sc-43009-SH, Santa Cruz Biotechnology) or Cs shRNA and Cdh23 shRNA to knockdown the transcription levels of Cs and Cdh23 genes.

Techniques: Expressing, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Transfection, shRNA, Knockdown, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction