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

Journal: iScience

Article Title: HHV-6B ribonucleotide reductase sequesters NF-κB subunit p65 to inhibit innate immune responses

doi: 10.1016/j.isci.2024.111710

Figure Lengend Snippet:

Article Snippet: Plasmid: pcDNA3-HA-human NEMO , Tang et al., , Addgene plasmid #13512.

Techniques: Virus, Recombinant, Protease Inhibitor, Reporter Assay, Isolation, Infection, Construct, shRNA, Expressing, Plasmid Preparation, Software, Imaging, Microscopy, Fluorescence

Journal:

Article Title: IQGAP1 Plays an Important Role in the Invasiveness of Thyroid Cancer

doi: 10.1158/1078-0432.CCR-10-1627

Figure Lengend Snippet: SW1736, KAT18 and FTC133 cells, at a density of 2.5×104 cells/well, were seeded on Matrigel invasion chamber plates and cultured in routine medium for 22 hr. Invasive cells that penetrated through pores and migrated to the underside of the membrane were stained by crystal violet and photographed under a microscope. A, Representative results on the invasion assay of three thyroid cancer cells. B, Histogram plots of the number of invading cells corresponding to A for each cell type. “C”, cell transfection with shRNA containing scrambled nucleotides; “S”, cell transfection with specific shRNA to knock down IQGAP1 or IQGAP3. (**P < 0.01)

Article Snippet: The annealed double-stranded oligonucleotides were cloned into the HpaI and XhoI sites of shRNA lentiviral expressing vector pSicoR-PGK-puro (Addgene Corp., Cambridge, MA) ( 27 ) and confirmed by sequencing.

Techniques: Cell Culture, Membrane, Staining, Microscopy, Invasion Assay, Transfection, shRNA, Knockdown

Journal:

Article Title: IQGAP1 Plays an Important Role in the Invasiveness of Thyroid Cancer

doi: 10.1158/1078-0432.CCR-10-1627

Figure Lengend Snippet: SW1736 and FTC133 cells stably transfected with control shRNA or specific shRNA to knock down IQGAP1 or IQGAP3 were seeded in six-well plates in 0.3% agar over a bottom layer of 0.6% agar and treated with routine medium plus 1.5 mg/ml puromycin. After 2–3 weeks, cell colony numbers were counted under microscope and cell colonies were photographed. A, Representative results of colony formation of SW1736 and FTC133 cells. B, Histogram plots of colony numbers corresponding to A for each cell type. Only cell colonies containing more than 50 cells were counted. KAT18 cells did not grow on soft agar. “C”, cell transfection with shRNA containing scrambled nucleotides; “S”, cell transfection with specific shRNA to knock down IQGAP1 or IQGAP3. (*P<0.05; **P<0.01)

Article Snippet: The annealed double-stranded oligonucleotides were cloned into the HpaI and XhoI sites of shRNA lentiviral expressing vector pSicoR-PGK-puro (Addgene Corp., Cambridge, MA) ( 27 ) and confirmed by sequencing.

Techniques: Stable Transfection, Transfection, Control, shRNA, Knockdown, Microscopy

Journal:

Article Title: IQGAP1 Plays an Important Role in the Invasiveness of Thyroid Cancer

doi: 10.1158/1078-0432.CCR-10-1627

Figure Lengend Snippet: Thyroid cancer cell lines with stable transfections were lysed for western blotting after 24 hr of serum starvation as described in Fig S1. Shown in Fig 4A is the Western blotting analysis of the levels of IQGAP1, E-cadherin, cdc42, phosphorylated FAK (pFAK), phosphorylated Src (pSrc), and β-actin before and after IQGAP1 knockdown. Specific and virtually complete knockdown of IQGAP1 was seen in all the three cells. All other indicated proteins were expressed in all the cells except for E-cadherin that was only expressed in SW1736 cells. An interesting up-regulation of E-cadherin after the knockdown of IQGAP1 was seen in SW1736 cells. Shown in Fig 4B is the immunoprecipitation of cell lysates from normal wild-type SW1736 cells using anti-IQGAP1 antibodies, followed by Western blotting of the immunoprecipitates with antibodies against the indicated proteins. Specific co-immunoprecipitation of E-cadherin, but not cdc42, with IQGAP1 was seen. This only occurred with specific monoclonal anti-IQGAP1 antibodies but not non-specific control mouse IgG. “C”, cell transfection with shRNA containing scrambled nucleotides; “S”, cell transfection with specific shRNA to knock down IQGAP1. “Input” using 1/100 of the amount of lysates used for immunoprecipitation was directly run for immunoblotting analysis as control to show the proteins of interest.

Article Snippet: The annealed double-stranded oligonucleotides were cloned into the HpaI and XhoI sites of shRNA lentiviral expressing vector pSicoR-PGK-puro (Addgene Corp., Cambridge, MA) ( 27 ) and confirmed by sequencing.

Techniques: Transfection, Western Blot, Knockdown, Immunoprecipitation, Control, shRNA