Journal: Annals of clinical and translational neurology

Article Title: Clinical heterogeneity within the ALS-FTD spectrum in a family with a homozygous optineurin mutation.

doi: 10.1002/acn3.52075

Figure Lengend Snippet: Figure 4. (A–C): Rt-PCR revealed OPTN-mRNA expression in PBMCs from the brother of the index patient (listed as Pat2, A). On a protein level, optineurin could not be detected in PMBC lysates (B) or lysates from postmortem frontal cortex of the index patient (listed as Pat1) in western blot analysis (C). (D–I) Anti-phospho-TDP-43 immunohistochemistry on spinal cord sections of a sporadic case of ALS (D–F) and of the index patient ALS and the optineurin mutation (G–I). Phospho-TDP43-positive neuronal skein-like and compact cytoplasmic inclusions in motor neurons in the control case (D) are also immunopositive (arrows) for two different anti-optineurin antibodies (E: non-C-terminal; F: C-terminal). In contrast, in the patient with the optineurin mutation, the inclusions (G) do not immunoreact with any of the two anti-optineurin antibodies (H and I). Scale bars: 50 lm: D–F; 100 lm: G–I;

Article Snippet: The Trans-Blot TurboTM Transfer System was used for transfer of proteins onto nitrocellulose membranes at 25 V for 15 min. Membranes with loaded proteins were incubated with EveryBlot Blocking Buffer (Bio-Rad) at room temperature for 30–60 min, followed by incubation with primary antibodies at 4°C for at least 8 h. We used two OPTN polyclonal antibodies (Proteintech, Cat. No. 10837-1-AP, at a dilution of 1:10,000 & Cayman, Item No. 100000, at a dilution of 1:1000), of which one binds to the C-terminus of optineurin (Cayman), and a GAPDH antibody (Cell Signaling, Cat No. 2118, at a dilution of 1:1000).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemistry, Mutagenesis, Control