Journal: Human molecular genetics

Article Title: Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

doi: 10.1093/hmg/ddac211

Figure Lengend Snippet: Figure 2. USH1C/harmonin expression in retinal cells. (A) Bulk RNA-seq of human RNs and MGCs. In RNs (filled bars) and MGCs (empty bars), USH1C/harmonin transcripts are detectable. (B) Western blot analysis of harmonin protein expression in human retina with affinity-purified polyclonal antibodies against harmonin (H3) showed two major bands at 64 and 70 kDa, which comigrate with harmonin_a1 in C. Lower bands may represent harmonin_c isoforms. (C) His-tagged harmonin a1 and SF-tagged harmonin b3 were transiently expressed in HEK293T cells. Pan antiharmonin H3 detected bands that co-migrate at the molecular weights of recombinant harmonin_a1 and harmonin_b3. Lower bands represent degraded products of harmonin_a and/or b. (D) Western blot analysis to validate the specificity of the harmonin antibody. A strong harmonin band is detected in HEK293T cells transfected with HA-tagged harmonin (Harm_a1_HA) or co-transfected with Harm_a1_HA and control siRNA (NTC). Harmonin is not detected in cells co-transfected with HA-tagged harmonin_a1 (Harm_a1_HA) and siHarm, indicating the specificity of the harmonin antibody. ∗∗∗P-value < 0.001. (E) Antiharmonin (H3) western blot of MGCs and neurons including photoreceptors cells (RNs) isolated from human retina. Overall harmonin is higher expressed in MGCs, but a more prominent band is found at 70 kDa in RNs. Western blot verification with antiglutamate synthetase (Glul), a common marker for MGCs, indicated <10% contamination of the RN fraction with MGCs when quantified. Western blot for pyruvate dehydrogenase E1-β (Pdhb) served as the loading control, indicating that 1.5-fold more protein was loaded of the RN fraction when quantified. (F) Localization of harmonin in retina sections. Indirect immunofluorescence labeling of harmonin in a longitudinal section through the retina, the OS, the IS and the nuclei in the ONL and MGC of a NHP and human retina, respectively. In addition to the prominent labeling of the OLM (arrowhead), patchy harmonin staining was present in the layer of the photoreceptor OS. Faint staining was present in the IS, the OPL, the IPL and GCL. Scale bars: 10 μm.

Article Snippet: Primary antibodies used were as follows: anti-Arl13b (ab136648, Abcam), anti-JAM-B (SAB2501282, Sigma-Aldrich), anti-β-catenin (cs-7963, Santa Cruz), anti-filamin A (67133-1-ig, Proteintech) and anti-arrestin (SCT128, Santa Cruz Biotechnology), antihis (27-4710-01, AmershamTM), anti-actin (MA5-11869, Thermo Fisher Scientific), anti-GFP (gift from Clay Smith), anti-pyruvate dehydrogenase E1-β (Pdhb) (ab155996, Abcam), anti-glutamine synthetase (Glul) (MAB302, Merck Millipore) and anti-RFP (6G6, Chromotek).

Techniques: Expressing, RNA Sequencing, Western Blot, Affinity Purification, Recombinant, Transfection, Control, Isolation, Marker, Immunofluorescence, Labeling, Staining