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: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: GFI1 enhances NF-κB and STAT3 activity by inhibiting SOCS1 expression in esophageal squamous cell carcinoma cell lines. (A and B) siGFI1 was transfected into KYSE30 and KYSE150 cells. (A) mRNA and (B) protein expression levels of SOCS1 were examined using reverse transcription-quantitative PCR and western blotting, respectively. (C) NF-κB activity, (D) p-p65 and (E) p-STAT3 protein levels were analyzed in KYSE30/KYSE150 cells following transfection with siGFI1. * P<0.05, ** P<0.01 and *** P<0.001 vs. control. si, small interfering RNA; GFI1, growth factor-independent 1; SOCS1, suppressor of cytokine signaling 1; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Activity Assay, Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: SOCS1 inhibits the increase in esophageal squamous cell carcinoma cell proliferation and migration induced by GFI1. (A-H) siSOCS1 was transfected into the GFI1-knockdown KYSE30 and KYSE150 cells. (A) Colony formation assays were performed on the double transfected cells. (B) Cell migration was determined using Transwell assays. (C) NF-κB activity, (D) p-p65, p-STAT3 and SOCS1 protein levels were analyzed. (E-G) Ratio of p-p65 to p65 and STAT3 to p-STAT3, as well as (H) the semi-quantification of SOCS1 protein expression were determined using ImageJ. Magnification, ×200. * P<0.05, ** P<0.01 *** P<0.001 vs. control; # P<0.05, ## P<0.01 and ### P<0.001 vs. siGFI1. SOCS1, suppressor of cytokine signaling 1; si, small interfering RNA; GFI1, growth factor-independent 1; p-, phosphorylated.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Migration, Transfection, Knockdown, Activity Assay, Expressing, Control, Small Interfering RNA

Journal: International Journal of Molecular Medicine

Article Title: GFI1 promotes the proliferation and migration of esophageal squamous cell carcinoma cells through the inhibition of SOCS1 expression

doi: 10.3892/ijmm.2021.5017

Figure Lengend Snippet: Regulatory model of the role of GFI1 in ESCC progression. The results of the present study demonstrated that the upregulation of GFI1 promoted ESCC proliferation and migration via inhibition of SOCS1 expression, leading to the activation of the NF-κB and STAT3 pathways. GFI1, growth factor-independent 1; ESCC, esophageal squamous cell carcinoma; SOCS1, suppressor of cytokine signaling 1.

Article Snippet: Rabbit polyclonal antibodies against phosphorylated (p)-p65 (1:1,000; cat. no. 310012) were obtained from Chengdu Zen Bioscience Co., Ltd. Rabbit monoclonal antibodies against STAT3 (1:1,000; cat. no. 4904S) and p-STAT3 (1:1,000; cat. no. 4113S) were purchased from Cell Signaling Technology, Inc.

Techniques: Migration, Inhibition, Expressing, Activation Assay

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 1. Inhibition of neuroblastoma cell lines employing crizotinib and PF-06463922. Treatment of neuroblastoma cells with the ALK TKIs crizotinib and PF-06463922. (A,B) Proliferation was assessed over 5 days using the resazurin cell proliferation assay in the following neuroblastoma cell lines: CLB-BAR, CLB-GE, CLB-PE, SK-N-AS and IMR32. Neuroblastoma cell line characteristics: CLB-GE – MYCN amplified, 1p deletion, 17q gain, amplified ALK amplicon containing an ALKF1174V mutation; CLB-BAR – amplified MYCN/ALK, Δexon 4-11, 1p deletion, 17q gain; IMR32 – MYCN, wt sequence but exons 2-4 are amplified, 1p deletion, 17q gain; CLB-PE – 1p gain, amplified MYCN, 17q gain; SK-N-AS – IGF-1 overexpressing, 1p deletion, 1q gain, 17q gain, 17 deletion. Cells lines were treated with increasing concentrations of either PF-06463922 (A) or crizotinib (B) as indicated. Data are mean±s.d. of the fold-relative fluorescence from treated cells relative to untreated cells from three independent experiments. (C) CLB-BAR, CLB-GE neuroblastoma cells were treated for 6 h with either crizotinib or PF-04643922 as indicated. Cellswere harvested andpre-clearedcelllysates were analyzed onSDS PAGE followedbywesternblotting for ALK, phospho-ALK-Y1278,phospho-ERK5, pan-ERK5 phospho-ERK1/2 and pan-ERK. Actin was employed as a loading control. Protein band intensities were quantified by Image Studio Lite 3.1 and normalized to untreated samples.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Proliferation Assay, Amplification, Mutagenesis, Sequencing, Fluorescence, Control

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 3. Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ-32P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison, Inhibition, Mutagenesis, In Vitro, Kinase Assay, Incubation, Activation Assay, Generated

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 5. Efficacy of PF-06463922 in an ALKF1174 CLB-GE xenograft neuroblastoma model. 4.5×106 CLB-GE cells were injected into left shoulder subcutaneously. (A) Tumor growth curves represent the average volume of vehicle group and PF-06463922-treated group, P≤0.05 (n=5 for each group). (B) Average tumor weights in vehicle group and PF-06463922 group are displayed, P=0.02. (C) Average body weight on the day of sacrifice is shown, P>0.05. (D) Immunoblotting analysis of indicated proteins from tumors collected after 8 days of treatment with vehicle or PF-0646399 and relapsed tumors. Tumor lysates were harvested, pre-cleared and analyzed by western blotting for ALK, phospho-ALK-Y1278, MYCN, phospho-ERK1/2 and phospho-AKT. Pan-ERK and pan-Akt were employed as loading controls. (E) Immunohistochemical staining of tumors for Ki-76 as a measure of proliferation rate as indicated. Ki67-positive cells were counted manually per field of vision and quantitative results (n=15). Statistical analysis shows significant difference between vehicle and PF-06463922- treated group, P<0.002 using unpaired t-test. Data in all graphs presented as mean±s.d.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 6. Preclinical efficacy of PF-06463922 in a murine model of Th-ALKF1174L/MYCN-driven neuroblastoma. (A) Waterfall plots of tumoral response in Th-ALKF1174L/MYCN mice treated with vehicle, crizotinib (100 mg/kg, once daily) or PF-06463922 (10 mg/kg, twice daily). Each bar indicates percent change in the volume of an individual tumor, as assessed by T2-weighted MRI, on day 7 compared with day 0 of treatment. (B) Representative MRI of each treatment group on day 0 and day 7 after treatment of indicated vehicle or drug. Hashed white line indicates tumor border. (C) Immunoblot analysis of phosphorylated ALK (phospho-ALK-Y1278), total ALK, and MYCN of tumors treated for 2 days with vehicle or PF-06463922. GAPDH was employed as loading control. Arrow indicates pALK-Y1278. (D) Quantification of band intensity of MYCN relative to GAPDH (left) and ratio of band intensity of pALK over total ALK (right) comparing vehicle versus treated samples. (E) Meso Scale Discovery assay depicted as electrochemiluminescence signal of treated samples relative to the respective vehicle controls for total ALK, phospho-ALK-Y1568, and the ratio of pALK to ALK. (F) Representative H&E (top) and immunohistochemical images for Ki67 (bottom) of vehicle and treated samples. Quantification of overall percentage of area positive for Ki67 (right). Error bars represent s.d. between individual animals (per group: n=4 in A, n=5 in C-F). P-values equal unpaired t-test comparison between vehicle and treatment groups.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Control, Electrochemiluminescence, Immunohistochemical staining, Comparison

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

Journal: Cell

Article Title: Inflammation Improves Glucose Homeostasis Through IKKβ-XBP1s Interaction

doi: 10.1016/j.cell.2016.10.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-phospho-XBP1s Thr48 , Covance , Raised against CRAAGSEASGT(p)PQARKRQR.

Techniques: Recombinant, Plasmid Preparation, Expressing, Sequencing, Software

Journal: Communications Biology

Article Title: CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1

doi: 10.1038/s42003-024-06857-4

Figure Lengend Snippet: A Detection of lipid droplets by oil red O staining in pGCs and KGN cells treated with or without CTBP1-siRNA (control siRNA as si-NC) (upper panel), and in cells further transfected with CTBP1 cDNA or vector. Scale bars, 20 µm. B Quantification of lipid droplets in cells from A. C . Detection of lipid droplets by Nile red staining in cells pretreated with CTBP1 siRNA and further transfected with CTBP1 cDNA. Scale bars, 20 µm. D Quantification of Nile red/cell from ( C ). E Relative TG levels detected in of 293T cells, pGCs and KGN cells expressing control (si-NC)/CTBP1 siRNA (upper panel) and vector/CTBP1 cDNA (lower panel). F Signal network analyzed by the inbio-discover protocol from https://inbio-discover.com/#search to predict the interconnecting signal networks between proteins. G, H Expression of ACC1, FASN, CPT1A, and SREBP1 detected by WB in KGN cells overexpressing control or CTBP1 siRNA ( G ), and in cells expressing vector or CTBP1 cDNA following control/CTBP1 siRNA treatment ( H ). Quantification results are presented as mean ± SD and analyzed by using two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 are considered statistically significant. LD Lipid droplet, pGCs Primary granulosa cell, TG Triglyceride, WB Western blot.

Article Snippet: A total of 500 mg proteins was used to incubate with 10 μg of antibodies to CTBP1 (ab129181, Abcam or sc-398945, anti-mouse, Santa Cruz), SREBP1 (ab28481, anti-rabbit, Abcam / sc-365513, anti-mouse, Santa Cruz / SREBP1 (PA1-337, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser439/415) (PA5-99371, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser396/372) (PA5-105169, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser338/314) (PA5-105567, anti-rabbit, ThermoFisher) or normal rabbit/mouse IgG (negative control) at 4 °C for 14–16 h. Thereafter, the samples were incubated with Protein A/G PLUS-Agarose beads (Santa Cruz, sc-2003) at 4 °C for 5–6 h. The conjugated agarose beads containing antibody-antigen mixture were then washed with IP buffer at 4 °C for five times.

Techniques: Staining, Control, Transfection, Plasmid Preparation, Expressing, Two Tailed Test, Western Blot

Journal: Communications Biology

Article Title: CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1

doi: 10.1038/s42003-024-06857-4

Figure Lengend Snippet: A Lipid droplet detection by oil red O staining after EGF treatment for 24 h. Scale bars, 20 µm. B Quantification analysis of lipid droplets from A. C Lipid droplet formation determined by Nile red staining after EGF treatment for 24 h. Scale bars, 50 µm. D Quantification analysis of Nile red/cell from ( C ). E Relative TG levels detected in KGN and pGCs treated with EGF for 24 h. F Detection of FASN, ACC1 and SREBF1 by WB in KGN cells after EGF treatment. G, H Lipid droplets detected by oil red O staining ( G ) and quantification ( H ) in KGN cells expressing control/CTBP1 siRNA after treatment with 100 ng/mL EGF for 24 h. Scale bars, 20 µm. I . Detection of TG in pGCs and KGN cells expressing CTBP1 siRNA and treated with EGF. J – K Detection of lipid droplets by Nile red staining ( L ) and quantification ( K ) in KGN cells expressing control/CTBP1 siRNA after treatment with 100 ng/mL EGF for 24 h. L Detection of ACC1, FASN and SREBP1 in KGN cells from ( G ). The quantification results are presented as mean ± SD and analyzed using two-tailed t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 are considered significant. LD, lipid droplet; pGCs, primary granulosa cell; WB, western blot.

Article Snippet: A total of 500 mg proteins was used to incubate with 10 μg of antibodies to CTBP1 (ab129181, Abcam or sc-398945, anti-mouse, Santa Cruz), SREBP1 (ab28481, anti-rabbit, Abcam / sc-365513, anti-mouse, Santa Cruz / SREBP1 (PA1-337, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser439/415) (PA5-99371, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser396/372) (PA5-105169, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser338/314) (PA5-105567, anti-rabbit, ThermoFisher) or normal rabbit/mouse IgG (negative control) at 4 °C for 14–16 h. Thereafter, the samples were incubated with Protein A/G PLUS-Agarose beads (Santa Cruz, sc-2003) at 4 °C for 5–6 h. The conjugated agarose beads containing antibody-antigen mixture were then washed with IP buffer at 4 °C for five times.

Techniques: Staining, Expressing, Control, Two Tailed Test, Western Blot

Journal: Communications Biology

Article Title: CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1

doi: 10.1038/s42003-024-06857-4

Figure Lengend Snippet: A mRNA levels of CTBP1 and SREBP1 mRNA detected by real time qPCR in KGN cells stably expressing vector or exogenous CTBP cDNA. B , C Semi-quantitative analysis of SREBP1a and SREBP1c showing that both pGCs and KGN cells express isoform SREBP1a other than SREBP1c. D Detection of pre-SREBP1, mature SREBP1, phosphorylated SREBP1, CTBP1 and FBXW7 in CTBP1 overexpressing KGN cells with or without MG-132 treatment, showing that SREBP1, phosphorylated SREBP1 at S396, and FBXW7 are increased following MG-132 treatment. E , F Co-IP and WB assays showing that overexpression of CTBP1 promotes SREBP1 high ubiquitination and lower binding to CTBP1. G – J Co-localization and fluorescent intensity of CTBP1 with SREBP1 ( G , H ) (Scale bars: 15 um) or with pS396-SREBP1 ( I – J ) (Scale bars: 10 um) in cells overexpressing CTBP1 with or without MG132 treatment.

Article Snippet: A total of 500 mg proteins was used to incubate with 10 μg of antibodies to CTBP1 (ab129181, Abcam or sc-398945, anti-mouse, Santa Cruz), SREBP1 (ab28481, anti-rabbit, Abcam / sc-365513, anti-mouse, Santa Cruz / SREBP1 (PA1-337, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser439/415) (PA5-99371, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser396/372) (PA5-105169, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser338/314) (PA5-105567, anti-rabbit, ThermoFisher) or normal rabbit/mouse IgG (negative control) at 4 °C for 14–16 h. Thereafter, the samples were incubated with Protein A/G PLUS-Agarose beads (Santa Cruz, sc-2003) at 4 °C for 5–6 h. The conjugated agarose beads containing antibody-antigen mixture were then washed with IP buffer at 4 °C for five times.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Co-Immunoprecipitation Assay, Over Expression, Binding Assay

Journal: Communications Biology

Article Title: CTBP1 links metabolic syndrome to polycystic ovary syndrome through interruption of aromatase and SREBP1

doi: 10.1038/s42003-024-06857-4

Figure Lengend Snippet: CTBP1 expression can be suppressed by FSH/LH, but induced by EGF through binding to their receptors. CTBP1 not only blocks the transcription of CYP19A1 to suppress aromatase expression and E2 synthesis, but also promotes the ubiquitination-mediated protein degradation of the nuclear pS396-SREBP1 to induce metabolic syndrome including abnormal lipid droplet formation, triglyceride and lipid synthesis.

Article Snippet: A total of 500 mg proteins was used to incubate with 10 μg of antibodies to CTBP1 (ab129181, Abcam or sc-398945, anti-mouse, Santa Cruz), SREBP1 (ab28481, anti-rabbit, Abcam / sc-365513, anti-mouse, Santa Cruz / SREBP1 (PA1-337, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser439/415) (PA5-99371, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser396/372) (PA5-105169, anti-rabbit, ThermoFisher), Phospho-SREBP1 (Ser338/314) (PA5-105567, anti-rabbit, ThermoFisher) or normal rabbit/mouse IgG (negative control) at 4 °C for 14–16 h. Thereafter, the samples were incubated with Protein A/G PLUS-Agarose beads (Santa Cruz, sc-2003) at 4 °C for 5–6 h. The conjugated agarose beads containing antibody-antigen mixture were then washed with IP buffer at 4 °C for five times.

Techniques: Expressing, Binding Assay