Journal: EMBO Molecular Medicine

Article Title: Parkin is a disease modifier in the mutant SOD 1 mouse model of ALS

doi: 10.15252/emmm.201808888

Figure Lengend Snippet: A, B Representative Western blot (A) and quantification (B) of p62 associated with mitochondria from spinal cords. Protein levels are normalized by subunit ATPase β of mitochondria (Complex V). Mitochondrial p62 levels are increased at 120 days (symptomatic stage) in SOD1‐G93A spinal cord relative to Non Tg and wild‐type SOD1 (wtSOD1). Results are expressed as mean ± SEM relative to Non Tg at 30 days; n = 8 mice (four males and four females). At 120 days, ** P = 0.0018 (Non Tg vs. SOD1‐G93A) and ** P = 0.0011 (SOD1‐G93A vs. wtSOD1) by paired one‐way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one‐way ANOVA with Tukey's correction at 60 and 90 days). C, D Representative Western blot (C) and quantification (D) of Tim23 in homogenates from spinal cords using β‐actin as normalizer. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n = 6 mice (three males and three females). *** P = 0.0002 (Non Tg vs. SOD1‐G93A at 120 days) by paired one‐way ANOVA with Tukey's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 days and paired one‐way ANOVA with Tukey's correction at 60 and 90 days). Tim23 is decreased in SOD1‐G93A spinal cord relative to Non Tg at 120 days. E, F Western blot (E) and quantification (F) of COXI in homogenates from spinal cords. Protein levels are normalized by β‐actin. Results are expressed as mean ± SEM and relative to Non Tg at 30 days; n = 6 mice (three males and three females). * P = 0.017 (Non Tg vs. SOD1‐G93A at 120 days) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found (paired Friedman's test with Dunn's correction at 30 and 60 days and paired one‐way ANOVA with Tukey's correction at 90 days). COX1 is decreased in SOD1‐G93A spinal cord relative to Non Tg at 120 days. G qPCR (fold change) of PGC1α mRNA normalized by β‐actin mRNA. Results are expressed as mean ± SEM and fold change of Non Tg at 30 days; n = 6 (three males and three females) for 30 and 120 days, n = 4 (two males and two females) for 60 days and n = 5 (three males and two females) for 90 days. * P = 0.035 at 90 days and *** P = 0.0007 at 120 days. Paired Student's t ‐test (for 60, 90, and 120 days) and paired Wilcoxon's test (for 30 days) were used for comparisons. Source data are available online for this figure.

Article Snippet: The following antibodies were used in Western blot (additional information can be found in ): Parkin (clone PRK8, 1:1,000, cat. # sc‐32282, Santa Cruz Biotechnology; Dallas, TX), β‐actin (clone AC‐74, 1:1,000–1:5,000, cat. # A5316, Sigma), p62 (clone 2C11, 1:1,000, cat. # H00008878‐M01, Novus Biologicals; Littleton, CO), optineurin (OPTN, 1:1,000, cat. # 10837‐1‐AP, Proteintech; Rosemont, IL), Complex V (ATP synthase beta, clone 3D5AB1, 1:1,000–1:5,000, cat. # A21351, Thermo Fisher Scientific; Waltham, MA), Tim23 (clone 32/Tim23, 1:1,000, cat. # 611222, BD Transduction Labs; San Jose, CA), COX1 (Complex IV, subunit 1, clone 1D6E1A8, 1:2,000, cat. #459600, Thermo Fisher Scientific), SOD1 (1:1,000, cat. # 574597, EMD Millipore; Billerica, MA), VCP (valosin‐containing protein, clone 5, 1:1,000, cat. # MA3‐004, Thermo Scientific; Rockford, IL), LC3 (microtubule‐associated protein 1A/1B‐light chain 3B, LC3B, 1:1,000, cat. # L7543, Sigma), March5 (membrane‐associated ring‐CH‐type finger 5, 1:1,000, cat. # PA5‐25584, Thermo Fisher Scientific), Mul1 (mitochondrial E3‐ubiquitin protein ligase 1 1:1,000, cat. # HPA017681, Sigma), Miro1 (mitochondrial Rho GTPase 1, clone 4H4, 1:1,000, cat. # H00055288‐M01, Novus Biologicals), mitofusin 2 (Mfn2, 1:1,000, cat. # M6319, Sigma), PARIS (parkin‐interacting substrate (ZNF746), clone N196/16, 1:1,000, cat. # 75‐195, NeuroMabs and Antibodies Inc.; Davis, CA), ubiquitin Lys48‐specific (clone Apu2, 1:1,000, cat. # 05‐1307, EMD Millipore), ubiquitin Lys63‐specific (clone Apu3, 1:1,000, cat. # 05‐1308, EMD Millipore), and horseradish‐peroxidase (HRP) goat anti‐rabbit, goat anti‐mouse, and donkey anti‐sheep secondary antibodies (1:10,000, Jackson ImmunoResearch; West Groove, PA).

Techniques: Western Blot

Journal: EMBO Molecular Medicine

Article Title: Parkin is a disease modifier in the mutant SOD 1 mouse model of ALS

doi: 10.15252/emmm.201808888

Figure Lengend Snippet: A, B Western blots of COXI (A) and Tim23 (B) in spinal cord homogenates at 130 days. C Quantification of COXI at 130 days with β‐actin as a normalizer. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.493 by paired Student's t ‐test). D Quantification of Tim23 at 130 days, using β‐actin as loading control, showed decreased levels of Tim23 in G93A mice. Results are expressed as mean ± SEM and as a percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.921 by paired Student's t ‐test). * P = 0.035 (Non Tg Vs. G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found. E, F Representative Western blots of COXI (E) and Tim23 (F) in disease end‐stage homogenates. G Quantification of COXI at disease end stage using β‐actin as loading control. Results are expressed as mean ± SEM and as percent of Non Tg; n = 5 (three males and two females) mice per group. * P = 0.047 for (G93A and PKO/G93A) by paired Student's t ‐test; ** P = 0.0018 by paired Friedman's test with Dunn's correction (Non Tg vs. G93A). H Quantification of Tim23 with β‐actin as normalizer at end stage showed that Parkin knockout can alleviate the turnover of mitochondrial proteins in G93A spinal cords. Results are expressed as mean ± SEM and as percent of Non Tg; n = 8 (four males and four females) mice per group. No statistically significant differences were found between G93A and PKO/G93A ( P = 0.190 by paired Student's t ‐test). * P = 0.037 (for PKO vs. PKO/G93A) by paired Friedman's test with Dunn's correction. No other statistically significant differences were found. Source data are available online for this figure.

Article Snippet: The following antibodies were used in Western blot (additional information can be found in ): Parkin (clone PRK8, 1:1,000, cat. # sc‐32282, Santa Cruz Biotechnology; Dallas, TX), β‐actin (clone AC‐74, 1:1,000–1:5,000, cat. # A5316, Sigma), p62 (clone 2C11, 1:1,000, cat. # H00008878‐M01, Novus Biologicals; Littleton, CO), optineurin (OPTN, 1:1,000, cat. # 10837‐1‐AP, Proteintech; Rosemont, IL), Complex V (ATP synthase beta, clone 3D5AB1, 1:1,000–1:5,000, cat. # A21351, Thermo Fisher Scientific; Waltham, MA), Tim23 (clone 32/Tim23, 1:1,000, cat. # 611222, BD Transduction Labs; San Jose, CA), COX1 (Complex IV, subunit 1, clone 1D6E1A8, 1:2,000, cat. #459600, Thermo Fisher Scientific), SOD1 (1:1,000, cat. # 574597, EMD Millipore; Billerica, MA), VCP (valosin‐containing protein, clone 5, 1:1,000, cat. # MA3‐004, Thermo Scientific; Rockford, IL), LC3 (microtubule‐associated protein 1A/1B‐light chain 3B, LC3B, 1:1,000, cat. # L7543, Sigma), March5 (membrane‐associated ring‐CH‐type finger 5, 1:1,000, cat. # PA5‐25584, Thermo Fisher Scientific), Mul1 (mitochondrial E3‐ubiquitin protein ligase 1 1:1,000, cat. # HPA017681, Sigma), Miro1 (mitochondrial Rho GTPase 1, clone 4H4, 1:1,000, cat. # H00055288‐M01, Novus Biologicals), mitofusin 2 (Mfn2, 1:1,000, cat. # M6319, Sigma), PARIS (parkin‐interacting substrate (ZNF746), clone N196/16, 1:1,000, cat. # 75‐195, NeuroMabs and Antibodies Inc.; Davis, CA), ubiquitin Lys48‐specific (clone Apu2, 1:1,000, cat. # 05‐1307, EMD Millipore), ubiquitin Lys63‐specific (clone Apu3, 1:1,000, cat. # 05‐1308, EMD Millipore), and horseradish‐peroxidase (HRP) goat anti‐rabbit, goat anti‐mouse, and donkey anti‐sheep secondary antibodies (1:10,000, Jackson ImmunoResearch; West Groove, PA).

Techniques: Western Blot, Knock-Out

Journal: Nature cell biology

Article Title: LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

doi: 10.1038/s41556-021-00672-3

Figure Lengend Snippet: a . A375 shFluc or shLIMIT cells were treated with IFNγ for 24 hours. RNA levels of LIMIT were determined by qRT-PCR. P value by 2-sided t-test. b . A375 shFluc or shLIMIT cells were treated with IFNγ for the indicated time. Protein levels of phospho-STAT1 (p-Y701), STAT1, and GAPDH were determined by Western blotting. 1 of 2 experiments is shown. c . A375 shFluc or shLIMIT cells were treated with IFNγ for 48 hours. Surface expression of HLA-ABC were determined by flow cytometry (FACS). P value by 2-sided t-test. d-g . YUMM1.7 ( d, e ) or CT26 ( f, g ) cells carrying shFluc or shLimit were treated with IFNγ. RNA levels of Limit were determined 24 hours after treatment ( d, f ). Surface staining of MHC-I (H2-D b ) was detected 48 hours after treatment ( e, g ). P value by 2-sided t-test. h-i . A375 WT or LIMIT promoter deletion cells were treated with IFNγ. RNA levels of LIMIT were determined 24 hours after treatment ( h ). Surface expression of HLA-ABC were determined 48 hours after treatment ( i ). P value by 2-sided t-test. j-k . B16 cells were transfected with dCas9-VPR, together with non-targeting sgRNA (sgNT) or sgRNA targeting the promoter of Limit (sgLimit). RNA levels of Limit were determined 24 hours after treatment ( j ). Surface expression of MHC-I (H2-D b ) or PD-L1 were determined 48 hours after treatment ( k ). P value by 2-sided t-test. l . B16-OVA cells carrying shFluc or shB2m were manipulated with Limit CRISPRa (sgLimit) for 48 hours. Surface expression of OVA-H2K b were determined by FACS. P value by 2-sided t-test. m-n . B16-OVA cells were manipulated with B2m knocking down (shB2m) or Limit CRISPRa (sgLimit), and co-cultured with OT-I cell at the ratio of 1:4. Cell death was measured by PI staining in CD45 − tumor cells. Dot plots ( m ) and statistical results ( n ) are shown. P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in ( a, c, d, e, f, g, h, i, j, k, l, n ). Source data are provided in Soure_data_Fig2.xlsx and Unmodified_blots_Fig2.pdf.

Article Snippet: For human MHC-I Western blotting, total cell lysates were denatured in the sample buffer without β-ME (non-reducing) to maintain the disulphide bridges and the conformation of the proteins to be detected by anti-HLA-ABC antibody (W6/32, Novus Biologicals, 64775, 1: 1,000).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry, Staining, Transfection, Cell Culture

Journal: Nature cell biology

Article Title: LIMIT is an immunogenic lncRNA in cancer immunity and immunotherapy

doi: 10.1038/s41556-021-00672-3

Figure Lengend Snippet: a-c . A375 cells were forced expression of GBPs. MHC-I RNA ( a ), surface expression ( b ) or total protein ( c ) levels were determined 24 hours ( a ) or 48 hours ( b , c ) afterwards. P value by 2-sided t-test. 1 of 2 blots is shown. d . MHC-I surface expression in YUMM1.7 or B16 cells upon Gbp2 overexpression. P value by 2-sided t-test. e . HSF1 chromatin IP for indicated gene promoters were performed in IFNγ-pretreated A375 cells. P value by 2-sided t-test. f . A375 cells were treated with 17-AAG to activate HSF1. Protein levels of HLA-ABC were determined 48 hours after treatment. 1 of 2 experiments is shown. g . A375 HSE-luc cells were forced expression of GBPs. Luciferase activity were detected 48 hours afterwards. P value by 2-sided t-test. h . A375 cells were forced expression of GBP1. Indicated proteins were determined 12 hours afterwards. 1 of 2 experiments is shown. i . A375 WT or GBPs KO cells were treated with IFNγ. Indicated proteins were determined 24 hours afterwards. 1 of 2 experiments is shown. j . A375 cells were forced expression of GBP1, and treated with KRIBB11. MHC-I surface expression was determined 48 hours afterwards. P value by 2-sided t-test. k-l . MC38 shFluc, shGbp2, shHsf1, or shGbp2+shHsf1 cells were treated with IFNγ. Indicated proteins ( k ) or MHC-I surface expression ( l ) were determined 48 hours afterwards. 1 of 2 experiments is shown. P value by 2-sided t-test. m . Tumor growth curves of MC38 shFluc, shGbp2, shHsf1, and shGbp2+shHsf1 cells in C57BL/6 mice. n = 5 animals, P value by 2-sided t-test for end point tumor volume. n . Percentages of intral-tumoral IFNγ + CD8 + T cells or TNFα + CD8 + T cells in MC38 tumors carrying shFluc, shGbp2, shHsf1, or shGbp2+shHsf1. n = 5 biological independent samples, P value by 2-sided t-test. All data are mean ± SD. n = 3 biological independent samples in ( a, b, d, e, g, j, l ). Source data are provided in Soure_data_Fig5.xlsx and Unmodified_blots_Fig5.pdf.

Article Snippet: For human MHC-I Western blotting, total cell lysates were denatured in the sample buffer without β-ME (non-reducing) to maintain the disulphide bridges and the conformation of the proteins to be detected by anti-HLA-ABC antibody (W6/32, Novus Biologicals, 64775, 1: 1,000).

Techniques: Expressing, Over Expression, Chromatin Immunoprecipitation, Luciferase, Activity Assay

Journal: Nature Communications

Article Title: Regulation of T-cell activation and migration by the kinase TBK1 during neuroinflammation

doi: 10.1038/ncomms7074

Figure Lengend Snippet: ( a ) IB analysis of phosphorylated (P-) AKT (Ser473), Foxo1 (Thr24), S6 (Ser235/236), S6K1 (Thr389), as well as their total protein controls in CD4 + T cells from WT and Tbk1 -TKO mice (6 weeks old). ( b ) IB analysis of the indicated phosphorylated (P-) and total proteins in CD4 + T cells activated by anti-CD3 plus anti-CD28 using a crosslinking method. ( c ) IB analysis of the indicated phosphorylated (P-) and total proteins in CD4 + T cells isolated from day 18 EAE-induced WT or Tbk1 -TKO mice. ( d ) AKT was isolated by IP from the lysates of thymocytes derived from WT or Tbk1 -TKO mice (6 weeks old), and the ubiquitinated AKT (Ub-AKT) was detected by IB using an anti-ubiquitin antibody detecting K48-linked polyubiquitin chains. ( e ) IB analysis using WT CD4 + T cells, pretreated for 1 h with a TBK1 inhibitor, MRT67307, of solvent control dimethylsulphoxide (DMSO) and subsequently stimulated for the indicated times with anti-CD3 plus anti-CD28 in the presence of a protein synthesis inhibitor, cycloheximide. ( f ) IB analysis of endogenous AKT and Actin and exogenous FLAG-TBK1 in whole-cell lysates of HEK293T cells, transfected as indicated and subsequently incubated for 2 h with DMSO or MG132 (25 μM). An arrowhead indicates the non-specific band. ( g ) IB analysis of the indicated exogenous proteins in the whole-cell lysates of HEK293 cells transfected with HA-AKT WT or the indicated point mutants either in the presence (+) or absence (−) of FLAG-tagged TBK1. An arrowhead indicates the non-specific band. ( h ) IB analysis of whole-cell lysates of WT CD4 + T cells transduced with pMIGR1 retroviruses encoding WT or mutant forms of AKT as well as the GFP marker. ( i ) IB (left) and flow cytometry (right) analyses of splenic CD4 + T cells from WT or Tbk1 -TKO mice treated injected (i.p) with rapamycin for 5 days ( n =5). ( j ) IB analysis of CD4 + cells from spleen of the indicated mouse strains. ( k , l ) EAE disease score ( k ) and CD4 + T-cell number of draining LN and CNS ( l , day27) ( l ) of the indicated mouse strains, immunized for EAE induction ( n =4). ( m ) Quantitative PCR (qPCR) analysis of the indicated genes in freshly isolated naïve or memory CD4 + T cells from WT and Tbk1 -TKO mice ( n =4). ( n ) S1PR1 flow cytometry analysis in memory CD4 + cell of inguinal LN from WT or Tbk1 -TKO mice. ( o ) qPCR analysis of KLF2 in CD4 + T cells treated for 1 h with either DMSO control or inhibitors for AKT (AKTi) or PI3 kinase (LY294002). Data represent mean±s.d. from two or more independent experiments. * P <0.05; ** P <0.01; NS, non-significant as determined by two-way analysis of variance with Bonferroni’s post test for EAE clinical scores analysis and two-tailed unpaired Student’s t -test for other analysis, comparing the indicated groups.

Article Snippet: Antibodies for phospho-TBK1 Ser172 (D52C2, 1:1,000), phospho-AKT1 Ser473 (D9E, 1:1,000), phospho-S6K1 Thr389 (1A5, 1:1,000), phospho-S6 Ser235/236 (D57.2.2E, 1:1,000), phospho-FoxO1 Thr24/FoxO3a Thr32 (1:1,000), phospho-p38 Thr180/Tyr182 (3D7, 1:1,000), TBK1, S6K1 (49D7, 1:1,000), S6 (54D2, 1:1,000) and FoxO1 (C29H4, 1:1,000) were from Cell Signaling Technology.

Techniques: Isolation, Derivative Assay, Ubiquitin Proteomics, Solvent, Control, Transfection, Incubation, Transduction, Mutagenesis, Marker, Flow Cytometry, Injection, Real-time Polymerase Chain Reaction, Two Tailed Test