Journal: Nature communications

Article Title: Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling.

doi: 10.1038/s41467-021-26477-4

Figure Lengend Snippet: Fig. 6 OPTN restrains the IL-6/IL-10 activated JAK2/STAT3 phosphorylation in DCs. a Expression of (p)-JAK2 in Ctrl or Optn deficient BMDCs stimulated with LPS for 8 h. b, c Ctrl and Optn deficient BMDCs were stimulated with LPS (b) or poly I:C (c) after transfection with empty or OPTN expression vector. The expressions of OPTN and p-JAK2 were confirmed by western blotting. d Western blotting for expression of (p)-JAK2 and (p)- STAT3 in Ctrl and Optn deficient BMDCs stimulated with anti-IL6 or anti-IL10 for 48 h. e Interaction between endogenous TLR4 and JAK2 or STAT3 in BMDCs was analyzed by IP with anti-JAK2 or anti-STAT3 using normal rabbit IgG as a control, followed by immunoblotting with anti-TLR4, anti-JAK2, and anti-STAT3. f Expression of (p-) signaling proteins in whole-cell lysates of Ctrl or Optn deficient BMDCs stimulated with LPS. g HEK293 cells were transfected with si-Optn or Optn plasmid before IL-6 stimulation. The expressions of OPTN and p-JAK2 were confirmed by western blotting. h Western blotting for expression of p-JAK2 and p-STAT3 in Ctrl and Optn deficient BMDCs stimulated with RAPA. All data are representative of three independent experiments. Source data are provided in Source data file.

Article Snippet: FLAG-tagged-JAK1, FLAG-tagged-JAK2, and FLAGtagged-JAK3 plasmids were obtained from OriGene.

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Nature communications

Article Title: Optineurin modulates the maturation of dendritic cells to regulate autoimmunity through JAK2-STAT3 signaling.

doi: 10.1038/s41467-021-26477-4

Figure Lengend Snippet: Fig. 7 OPTN inhibits the dimerization of JAK2 and subsequent STAT3 activation. a HA-OPTN expression plasmid was transfected with Flag-JAK1, Flag- JAK2, or Flag-JAK3 expression plasmid into HEK293 cells. Expression of OPTN and JAKs was confirmed by immunoblotting. Interaction between OPTN and JAKs was determined by immunoprecipitation (IP) with anti-HA antibody followed by immunoblotting with anti-Flag antibody. b Interaction between endogenous OPTN and JAK2 in BMDCs was analyzed by IP. c Schematic illustration of the truncated JAK2. Interactions between OPTN and truncation JAK2 in transiently transfected HEK293 cells were determined by IP. d Interactions between JAK2 with OPTN or OPTNΔUBD in transiently transfected HEK293 cells were determined by IP. e Interactions between HA-JAK2 and FLAG-JAK2 in transiently transfected HEK293 cells was determined by IP. f, g HEK293 cells were transfected with FLAG-JAK2 and HA-OPTN or empty vector (f). BMDCs were transfected with HA-OPTN or empty vector (g). Whole- cell lysates were incubated with disuccinimidyl suberate (DSS). JAK2 antibody marked two bands: the upper band referring to the dimer and the lower band representing the monomer of JAK2. h Interaction of FLAG-JAK2 and HA-STAT3 in transiently transfected HEK293 cells was determined by IP. i BMDCs were transfected with HA-OPTN or empty vector. Interaction between endogenous STAT3 and JAK2 in BMDCs was analyzed by IP. j ChIP-seq analysis of the binding between STAT3 and Il-10 or Il-6 from GSE27161. k Il-10 and Il-6 mRNA level in Ctrl and Optn deficient BMDCs stimulated with LPS for different times. n = 3 independent experiments, P(4) = 3.2E−06, P(8) = 0.0497, P(12) = 1.2E−05, P(16) = 3.9E−07, P(24) = 0.0060 of Il-10. Data in (a–i) are representative of three independent experiments. Data are presented as means ± SD. *P < 0.05; **P < 0.01; ***P < 0.001. Unpaired two-tailed Student’s t-test. Source data are provided in Source data file.

Article Snippet: FLAG-tagged-JAK1, FLAG-tagged-JAK2, and FLAGtagged-JAK3 plasmids were obtained from OriGene.

Techniques: Activation Assay, Expressing, Plasmid Preparation, Transfection, Western Blot, Immunoprecipitation, Incubation, ChIP-sequencing, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: NRVMs were transduced with AdV5-CMV-CRYAB R120G -GFP and treated with 3 µM ruxolitinib or DMSO or 100 nM siRNAs targeting either JAK1 (siJak1), JAK2 (siJak2), STAT3 (siStat3) or scramble siRNA (scr). Thereafter, NRVMs were harvested or fixed after 4-6 days. A , Treatment with ruxolitinib or DMSO; Scale bar = 100 µm. B , Transfection with siJak1; Scale bar = 200 µm. C , Transfection with siJak2; Scale bar = 100 µm D , Transfection with siStat3, Scale bar = 20 µm. A , B , C , D , Western blots of protein extracts from treated NRVMs were stained with antibodies directed against indicated proteins. In the representative immunofluorescence images, aggregates are depicted in magenta (CRYAB R120G -GFP), cardiomyocytes in yellow (anti-cardiac troponin I), and nuclei in blue (DAPI). Quantification of aggregates in cardiomyocytes with NIS Elements or ImageJ software. Data were obtained from 1 (Western blot) or 2 (immunofluorescence) NRVM preparations with at least 3 wells per condition and at least 7 images per well for immunofluorescence. Data are depicted as mean ± SEM, and p-values were obtained with the unpaired Student’s t-test. Abbreviation: ns, non-significant.

Article Snippet: After electrophoresis, the proteins were transferred to nitrocellulose or PVDF membranes with Bio-Rad Tris/Glycine buffer or self- made transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.3) and stained with the following antibodies: JAK1 – R&D systems, MAB4260; JAK2 – Cell Signaling Technology, 3230; STAT3 - Cell Signaling Technology, 12640; P-STAT3 – Cell Signaling Technology, 9145S; GAPDH – HyTest, 5G4 or Sigma-Aldrich, P0067; ACTN2 - Sigma- Aldrich, A7811; GFP – Santa Cruz Biotechnology, sc-9996; LC3B – Cell Signaling Technology, 2775; p70 S6K – Cell Signaling, 34475.

Techniques: Transduction, Transfection, Western Blot, Staining, Immunofluorescence, Software

Journal: bioRxiv

Article Title: Ruxolitinib clears CRYAB p.Arg120Gly aggregates through the ubiquitin-proteasome system

doi: 10.1101/2024.10.11.615348

Figure Lengend Snippet: Heterozygous (het) or homozygous (hom) Jak1 knockout (KO) was induced in MCM-transgenic (TG) mice crossed with CRYAB p.Arg120Gly TG (R120G) or non-transgenic (NTG) mice with tamoxifen chow. Transthoracic echocardiography was performed 26 weeks and hearts were extracted. A , Scheme of experimental outline. B , Jak1 mRNA levels determined by RT-qPCR. C , JAK1 protein levels determined by Western blot and normalized to ACTN2. Western blot quantification was performed with Image Lab software D, Ejection fraction (EF) and left ventricular mass-to-body weight ratio (LVM/BW). E , Heart weight-to-body weight ratio (HW/BW) and body weight (BW). F , Representative images and quantification of mouse heart sections. Aggregates are depicted in green, cardiomyocytes in purple (anti-cardiac troponin I) and nuclei in blue (DAPI). Scale bar = 50 µm. Quantification of aggregates in cardiomyocytes with NIS Elements software. At least 5 images of 3 mice per group were analyzed. Data are depicted as mean ± SEM, and p-values were obtained with the one-way ( B ) or two- way ANOVA ( D , E ) with Tukey’s multiple comparisons post-hoc analysis or unpaired Student’s t-test ( C , F ). Abbreviation: ns, non-significant.

Article Snippet: After electrophoresis, the proteins were transferred to nitrocellulose or PVDF membranes with Bio-Rad Tris/Glycine buffer or self- made transfer buffer (25 mM Tris base, 190 mM glycine, 20% methanol, pH 8.3) and stained with the following antibodies: JAK1 – R&D systems, MAB4260; JAK2 – Cell Signaling Technology, 3230; STAT3 - Cell Signaling Technology, 12640; P-STAT3 – Cell Signaling Technology, 9145S; GAPDH – HyTest, 5G4 or Sigma-Aldrich, P0067; ACTN2 - Sigma- Aldrich, A7811; GFP – Santa Cruz Biotechnology, sc-9996; LC3B – Cell Signaling Technology, 2775; p70 S6K – Cell Signaling, 34475.

Techniques: Knock-Out, Transgenic Assay, Quantitative RT-PCR, Western Blot, Software

Journal: Journal of Biological Chemistry

Article Title: Ultraviolet Radiation Inhibits Interleukin-2-induced Tyrosine Phosphorylation and the Activation of STAT5 in T Lymphocytes

doi: 10.1074/jbc.m006372200

Figure Lengend Snippet: FIG. 4. UV radiation does not inhibit IL-2-induced tyrosine phosphorylation of Jak1 and Jak3. CTLL cells were left untreated (lane 1), stimulated with 100 units/ml IL-2 (lane 2), irradiated with 400 J/m2 UV (lane 3), or treated with UV plus IL-2 (lane 4). Samples in lanes 5 and 6 were preincubated with 1 mM Na3VO4 for 2 h before stimulation with IL-2 (lane 5) or with UV plus IL-2 (lane 6). 15 min after stimulation immunoprecipitations were performed using antibodies directed against Jak1 or Jak3. Western blot analyses were performed using an antibody against phosphotyrosine (P-Tyr), followed by reprobing of the membranes with the respective anti-Jak antibodies.

Article Snippet: Immunoprecipitations were carried out with 1 ml of a 1 mg/ml protein solution supplemented with 5 mg of the corresponding antibody directed against Jak1 (AF602; R & D Systems), Jak3 (05-406; Upstate Biotechnology, Lake Placid, NY), or STAT5 (PA-ST5A; R & D Systems) and with 50 ml of protein A/Gagarose (Santa Cruz) at 4 °C overnight.

Techniques: Phospho-proteomics, Irradiation, Western Blot