Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) The expression of endogenous FAT10 was stimulated by treating HEK293 cells with TNF/IFNγ for 24 h, followed by immunoprecipitation (IP) with a monoclonal anti-FAT10 antibody (4F1), SDS–PAGE and Coomassie blue staining (left panel). Endogenous FAT10 was cut out and sent for a phospho-proteomic analysis. As a control, samples were additionally analyzed by immunoblotting (IB) (right panel). Endogenous FAT10 was visualized with a FAT10-reactive, rabbit polyclonal antibody . β-Actin was used as a loading control. Asterisks mark the heavy and light chains of the FAT10-reactive antibody used for the immunoprecipitation. (B) Ribbon diagram of FAT10 showing the phosphorylated amino acids Ser62, Ser64, Thr77, Ser95, and Ser109 in the N- (red) and C- (blue) ubiquitin-like domain, respectively. (C) Radiolabeled phosphate was incorporated into recombinant FAT10 or ubiquitin during incubation with the recombinant kinases PINK1 or MAPKAPK3 (MK3) at 30°C for 15 or 45 min. The autoradiogram shows substrate specificity of MAPKAPK3 and PINK1 to phosphorylate FAT10 and ubiquitin, respectively. One representative experiment out of three independent experiments with similar outcomes is shown. Source data are available for this figure.

Article Snippet: Antibodies used for immunoblotting are as follows: mouse anti-FLAG antibody (F1804, 1:3,000; Merck), mouse anti-FLAG (HRP) antibody (A8592, 1:3,000; Merck), rabbit anti-FLAG antibody (F7425, 1:750; Merck), mouse anti-HA antibody (H3663, 1:5,000; Merck), rabbit anti-HA antibody (H608, 1:1,000; Merck), rabbit anti-GAPDH antibody (G9545, 1:10,000; Merck), mouse anti-tubulin antibody (T6557, 1:10,000; Merck), rabbit anti-FAT10 antibody ([ ] 1:750), rabbit anti-MK3 antibody (3043, 1:1,000; Cell Signaling), rabbit anti-IKKβ antibody (2684, 1:1,000), rabbit anti-IRF3 antibody (11904, 1:1,000; Cell Signaling), rabbit anti-phospho-IRF3 antibody (4947, 1:1,000; Cell Signaling), rabbit anti-IKKε (3416, 1:1,000; Cell Signaling), rabbit anti-phospho-IKKε antibody (06-1340, 1:1,000; Merck), mouse anti-RIG-I antibody (MABF297, 1:1,000; Merck), mouse anti-OTUB1 antibody (CF505157, 1:1,000; Thermo Fisher Scientific), mouse anti-M1 antibody (ab22395, 1:1,000; Abcam), rabbit anti-TRAF3 antibody (ab239357, 1:1,000; Abcam), 800CW goat anti-mouse IgG (926-332210 1:10,000; Licor), and 680RD goat anti-rabbit antibody (926-68071, 1:10,000; Licor).

Techniques: Expressing, Immunoprecipitation, SDS Page, Staining, Control, Western Blot, Ubiquitin Proteomics, Recombinant, Incubation

Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) His-3xFLAG-FAT10 (FLAG-FAT10), HA-tagged MK3, HA-MK3 TT/EE (constitutively active mutant), or HA-MK3 TT/AA (inactive mutant), were transiently overexpressed in HEK293 cells for 24 h, followed by lysis and immunoprecipitation (IP) with a monoclonal phosphoserine-reactive antibody. Subsequently, an immunoblot (IB) was performed using the antibodies indicated. Where indicated, cells were stimulated with TNF/IFNγ for 24 h before harvesting and lysis. Moreover, cells were starved for 24 h (0.3% FCS DMEM) followed by TPA treatment (30 min) before lysis, where indicated. Calf intestinal alkaline phosphatase phosphatase was added to the lysates used in lanes 3 and 5, 2 h before performing the immunoprecipitation. (B) HEK293 cells were transiently transfected with an expression plasmid for FLAG-tagged FAT10 and where indicated, additionally treated with TNF/IFNγ for 24 h. Endogenous FAT10 expression was induced by treating HEK293 cells with TNF/IFNγ for 24 h. Where indicated, lysates were incubated with 400 U of λ phosphatase for 30 min at 30°C, before the immunoprecipitation was performed. Subsequently, an immunoprecipitation against FAT10 was performed using a monoclonal FAT10-reactive antibody (clone 4F1, ) coupled to protein A sepharose, followed by Phos-tag/SDS–PAGE and IB analysis with the antibodies indicated. γ-tubulin was used as loading control. (C) Cells were prepared as in (A) and treated as specified, followed by FLAG-IP, Phos-tag/SDS–PAGE, and IB analysis with the indicated antibodies. One representative example out of three independent experiments with similar outcomes is shown. Source data are available for this figure.

Article Snippet: Antibodies used for immunoblotting are as follows: mouse anti-FLAG antibody (F1804, 1:3,000; Merck), mouse anti-FLAG (HRP) antibody (A8592, 1:3,000; Merck), rabbit anti-FLAG antibody (F7425, 1:750; Merck), mouse anti-HA antibody (H3663, 1:5,000; Merck), rabbit anti-HA antibody (H608, 1:1,000; Merck), rabbit anti-GAPDH antibody (G9545, 1:10,000; Merck), mouse anti-tubulin antibody (T6557, 1:10,000; Merck), rabbit anti-FAT10 antibody ([ ] 1:750), rabbit anti-MK3 antibody (3043, 1:1,000; Cell Signaling), rabbit anti-IKKβ antibody (2684, 1:1,000), rabbit anti-IRF3 antibody (11904, 1:1,000; Cell Signaling), rabbit anti-phospho-IRF3 antibody (4947, 1:1,000; Cell Signaling), rabbit anti-IKKε (3416, 1:1,000; Cell Signaling), rabbit anti-phospho-IKKε antibody (06-1340, 1:1,000; Merck), mouse anti-RIG-I antibody (MABF297, 1:1,000; Merck), mouse anti-OTUB1 antibody (CF505157, 1:1,000; Thermo Fisher Scientific), mouse anti-M1 antibody (ab22395, 1:1,000; Abcam), rabbit anti-TRAF3 antibody (ab239357, 1:1,000; Abcam), 800CW goat anti-mouse IgG (926-332210 1:10,000; Licor), and 680RD goat anti-rabbit antibody (926-68071, 1:10,000; Licor).

Techniques: Mutagenesis, Lysis, Immunoprecipitation, Western Blot, Transfection, Expressing, Plasmid Preparation, Incubation, SDS Page, Control

Journal: Life Science Alliance

Article Title: FAT10 is phosphorylated by IKKβ to inhibit the antiviral type-I interferon response

doi: 10.26508/lsa.202101282

Figure Lengend Snippet: (A) HEK293 cells were transiently transfected with a His-3xFLAG-FAT10 (FLAG-FAT10) expression construct and stimulated for 24 h with TNF. Lysates were subjected to immunoprecipitation using FLAG-reactive antibodies, coupled to sepharose beads, and subsequently analyzed by Phos-tag/SDS–PAGE/IB analysis. Where indicated, cells were pretreated before TNF stimulation with the displayed inhibitors for a total of 3 h (10 µM each). (B) HEK293 cells were transiently transfected with expression plasmids for the different kinases. Cells were harvested, lysed, and subjected to immunoprecipitation using anti-FLAG or anti-HA antibodies, coupled to sepharose beads. Subsequently, the immunoprecipitated kinases were incubated with recombinant FAT10 (rFAT10) and an in vitro reaction was performed in the kinase buffer. The phosphorylation status of FAT10 was analyzed by Phos-tag/SDS–PAGE and IB. Asterisks mark unspecific background bands. (C) FLAG-FAT10 and the indicated kinases were transiently overexpressed in HEK293 cells followed by TNF stimulation. After 24 h, cells were lysed and subjected to immunoprecipitation against the FLAG-tag, combined with Phos-tag/SDS–PAGE and IB analysis. (D) Recombinant FAT10 (rFAT10) was incubated with recombinant kinases IKKβ, IKKε or MK3 for 45 min at 30°C. Subsequently, proteins were separated on a Phos-tag/SDS–PAGE followed by immunoblot analysis using the antibodies indicated. (E) HEK293 cells were prepared as described in (A). Where specified, cells were pretreated with the inhibitors indicated (10 µM each) for a total of 3 h before stimulation with TNF. One representative example out of three independent experiments with the same outcomes is shown. Source data are available for this figure.

Article Snippet: Antibodies used for immunoblotting are as follows: mouse anti-FLAG antibody (F1804, 1:3,000; Merck), mouse anti-FLAG (HRP) antibody (A8592, 1:3,000; Merck), rabbit anti-FLAG antibody (F7425, 1:750; Merck), mouse anti-HA antibody (H3663, 1:5,000; Merck), rabbit anti-HA antibody (H608, 1:1,000; Merck), rabbit anti-GAPDH antibody (G9545, 1:10,000; Merck), mouse anti-tubulin antibody (T6557, 1:10,000; Merck), rabbit anti-FAT10 antibody ([ ] 1:750), rabbit anti-MK3 antibody (3043, 1:1,000; Cell Signaling), rabbit anti-IKKβ antibody (2684, 1:1,000), rabbit anti-IRF3 antibody (11904, 1:1,000; Cell Signaling), rabbit anti-phospho-IRF3 antibody (4947, 1:1,000; Cell Signaling), rabbit anti-IKKε (3416, 1:1,000; Cell Signaling), rabbit anti-phospho-IKKε antibody (06-1340, 1:1,000; Merck), mouse anti-RIG-I antibody (MABF297, 1:1,000; Merck), mouse anti-OTUB1 antibody (CF505157, 1:1,000; Thermo Fisher Scientific), mouse anti-M1 antibody (ab22395, 1:1,000; Abcam), rabbit anti-TRAF3 antibody (ab239357, 1:1,000; Abcam), 800CW goat anti-mouse IgG (926-332210 1:10,000; Licor), and 680RD goat anti-rabbit antibody (926-68071, 1:10,000; Licor).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, SDS Page, Incubation, Recombinant, In Vitro, Phospho-proteomics, FLAG-tag, Western Blot

Journal: Biology Direct

Article Title: Analysis of state 1—state 2 transitions by genome editing and complementation reveals a quenching component independent from the formation of PSI-LHCI-LHCII supercomplex in Arabidopsis thaliana

doi: 10.1186/s13062-023-00406-5

Figure Lengend Snippet: Immunodecoration of thylakoid phosphoproteins with α-P-Thr and α-P-Ser antibodies. Isolated thylakoid samples corresponding to approximately 0.5 and 5 μg of Chl were blotted following separation on SDS PAGE and probed with α-P-Thr (panel A ) and α-P-Ser antibodies (panel B ), respectively. Experiments included all genotypes created in this work treated with PSII-favoring light and a dark-adapted (equivalent to state 1) wild type sample control. The pattern of the α-P-Thr reaction (panel A ) revealed the characteristic thylakoid phosphoproteins D1 (PsbA), D2 (PsbD) and LHCII. Consistent with the results presented in figure , the LHCII signal was extremely low in the dark-adapted wild type and was entirely missing in the koLhcb1 and kostn7 genotypes. kostn7 (and to lower extent the koLhcb2 and cB2.1 T40V lines) exhibited a stronger Thr phosphorylation of the PSII core complex subunits D1 and D2. Enhanced LHCII Thr phosphorylation was observed in the koLhcb2 and cB.1 T40V lines because of persistent plastoquinone reduction and the active state of the STN7 kinase towards the Lhcb1 Thr-38 residue. The α-P-Ser reaction (panel B ), instead, revealed an equal phosphorylation level of the LHCII band in all genotypes, except for the koLhcb1 sample where the faint reactive band corresponds to the phosphorylated serine(s) belonging to Lhcb2 polypeptides

Article Snippet: The global phosphorylation status of thylakoid proteins was assessed using α-phospho-threonine (Cell Signaling Technology, Danvers, USA, #9386) and α-phospho-serine (AS20 4487) antibodies.

Techniques: Isolation, SDS Page, Control, Phospho-proteomics, Residue