Journal: Nature Communications

Article Title: O -GlcNAc modification of leucyl-tRNA synthetase 1 integrates leucine and glucose availability to regulate mTORC1 and the metabolic fate of leucine

doi: 10.1038/s41467-022-30696-8

Figure Lengend Snippet: a SW620 cells were starved and stimulated with 11 mM glucose for the indicated durations. b Quantification of LARS1 S720 phosphorylation and O -GlcNAcylation of a (mean ± SEM, n = 3 independent experiments). c , d SW620 cells were transfected with the indicated siRNAs. After 48 h, the cells were starved of glucose for 4 h, pre-incubated with streptolysin O (SLO) for 5 min, and supplemented with 11 mM glucose or 200μM indicated metabolites for 30 min. e SW620 cells were transfected with the indicated siRNAs. After 48 h, the cells were starved of glucose for 4 h with vehicle or indicated compounds (10μM Compound C, 20 μM SBI-0206965, 1 μM LYN1604, 1 mM AICAR) f SW620 cells were transfected with the indicated LARS1 constructs. After 24 h, the cells were starved of glucose for 4 h and supplemented with 11 mM glucose for 30 min. g SW620 cells were transfected with control or siRNA targeting OGT1. After 48 h, the cells were starved of glucose at the indicated durations. h SW620 cells were transfected with siRNA targeting ULK1. After 24 h, the cells were transfected with the indicated expression construct. After 24 h, the cells were starved of glucose for 4 h. Each sample was analyzed by immunoblotting with the indicated antibodies. i SW620 control or S1042A knock-in cells were starved of glucose for 4 h and supplemented with glucose for 30 min. a , c , d , e , f , g , i Each sample was subjected to immunoprecipitation with sWGA-conjugated agarose beads, anti-LARS1 antibody-conjugated beads or anti-myc antibody-conjugated agarose beads and analyzed by immunoblotting with the indicated antibodies. Representative data of three experiments with similar results. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were obtained from the following sources: antibodies against phospho-p70 S6 kinase (T389)(WB dilution 1:1000, Cell signaling, #9205), p70 S6 kinase (WB dilution 1:1000, Cell signaling, #9202), phospho-4EBP1 (Thr37/46) (236B4) (WB dilution 1:1000, Cell signaling, #2855), 4EBP1 (53H11) (WB dilution 1:1000, Cell signaling, #9644), RagC (D8H5) (WB dilution 1:1000, Cell signaling, #9480), RagB (D18F3) (WB dilution 1:1000, Cell signaling, #8150), mTOR (7C10) (WB dilution 1:1000, Cell signaling, #2983), Raptor (24C12) (WB dilution 1:1000, Cell signaling, #2280), Hexokinase II (C64G5) (WB dilution 1:1000, Cell signaling, #2867), Aldolase A (D73H4) (WB dilution 1:1000, Cell signaling, #8060), DYKDDDDK Tag (Binds to same epitope as Sigma’s Anti-FLAG® M2 Antibody) (9A3) (WB dilution 1:1000, Cell signaling, #8146), phospho-AMPKα (T172), (40H9) (WB dilution 1:1000, Cell signaling, #2535), AMPKα (WB dilution 1:1000, Cell signaling, #2532), AMPKγ (WB dilution 1:1000, Cell signaling, #4187), phospho-ULK1 (S757) (WB dilution 1:1000, Cell signaling, #6888), phospho-Atg13 (S355) (D6J1W) (WB dilution 1:1000, Cell signaling, #26839), Atg13 (D4P1K) (WB dilution 1:1000, Cell signaling, #13273), phospho-Atg14 (S29) (D4B8M) (WB dilution 1:1000, Cell signaling, #92340), Atg14 (D1A1N) (WB dilution 1:1000, Cell signaling, #96752), LARS1 (WB dilution 1:1000, Cell signaling, #13868), RagD (WB dilution 1:1000, Bethyl Laboratories, #A304-301A), LARS1 (WB dilution 1:1000, IF dilution 1:200, IP: 2ug/400ug protein, Bethyl Laboratories, #A304-315A), LAMP2 (H4B4) (WB dilution 1:1000, IF dilution 1:50, Santa cruz, #c-18822), ARF1 (ARFS 1A9/5) (WB dilution 1:1000, Santa cruz, #sc-53168), b-actin (C4) (WB dilution 1:1000, Santa cruz, #sc-47778), c-Myc (9E10) (WB dilution 1:1000, IP: 2ug/300ug protein, Santa cruz #sc-40), O-GlcNAc (RL2), (WB dilution 1:1000, Santa cruz, #sc-59624), HA-Tag (F-7) (WB dilution 1:1000, Santa cruz, #sc-7392), ULK1 (H-240) (WB dilution 1:1000, Santa cruz, #sc-33182), OGT1 (WB dilution 1:1000, IP: 2ug/400ug protein, abcam #ab96718), phospho-PDCD4 (S67) (WB dilution 1:1000, abcam, #ab73343), PDCD4 (WB dilution 1:1000, abcam, #ab80590), Aldolase B (WB dilution 1:8000, Proteintech, #18065-1-AP), Aldolase C (WB dilution 1:5000, Proteintech, #A-11001), Sestrin 2 (WB dilution 1:2000, Proteintech, #10795-1-AP), EPRS1 (WB dilution 1:5,000, Neomics, #NMS-01-0004), Alexa488-conjugated secondary antibody (IF dilution 1:500 Invitrogen #14884-1-AP), Alexa594-conjugated secondary antibody (IF dilution 1:500 Invitrogen #A-11012), Anti-mouse IgG, HRP secondary antibody (WB dilution 1:10000 Invitrogen #31430), and anti-rabbit IgG, HRP secondary antibody (WB dilution 1:10000 Invitrogen #31460).

Techniques: Phospho-proteomics, Transfection, Incubation, Construct, Control, Expressing, Western Blot, Knock-In, Immunoprecipitation

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization.

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Figure 3. STAT3 is a direct binding target for isoquercitrin, with isoquercitrin specifically binding to the Ser668–Gln635–Gln633 site of STAT3. A) STAT3 in proteome microarray results. B) Pull-down experiments to detect the binding of isoquercitrin to STAT3 (n = 3). C) Surface plasmon resonance precisely measures the binding affinity and mode of isoquercitrin to STAT3. D) Cellular thermal shift assay (CETSA) confirms whether isoquercitrin can enter cells and bind to STAT3 (n = 3). E) Molecular docking and dynamics simulations assessing the binding mode of isoquercitrin with STAT3. F) The MM/GBSA method calculates the contributions of different amino acid residues in the isoquercitrin–STAT3 binding. The stability of the isoquercitrin–STAT3 binding was assessed through root-mean-square deviation (G), radius of gyration (H), and root-mean-square fluctuation (I). J) Alanine scan calculates the changes in affinity between isoquercitrin and STAT3 after mutating amino acid residues to alanine. The changes in the binding of isoquercitrin to STAT3 were detected using CETSA after mutating specific amino acids to alanine (K, L, M) (n = 3).

Article Snippet: Chemicals and Reagents: Antibodies specific for Phospho-STAT3Tyr705 (9145; 1:2000 for WB, 1:100 for immunofluorescence, 1:200 for immunohistochemistry), and STAT3 (4904; 1:2000 for WB) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay, Microarray, SPR Assay, Thermal Shift Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization.

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Figure 4. Isoquercitrin occupies the SH2 domain, inhibiting STAT3 phosphorylation, dimerization, and downregulating its transcriptional activity. A) Western blot (WB) detection of isoquercitrin effects on phosphorylated STAT3 (p-STAT3) and total STAT3 in kidneys (n = 4). B) Immunohistochemistry detection of p-STAT3 in glomerulus and kidney tubules (n = 6). Black arrows indicate p-STAT3 positive regions. C) Immunofluorescence co-staining of p- STAT3 and the endothelial cell marker Endomucin. White arrows indicate p-STAT3 positive regions. D) After crosslinking protein–protein interactions with DSS, the effect of isoquercitrin on STAT3 dimers in 293T cells was detected using WB. E) The determination of the half-maximal inhibitory concentration for the inhibition of STAT3 transcriptional activity by isoquercitrin. F) Isoquercitrin inhibits STAT3 phosphorylation and dimerization. Data are presented as the mean ± SEM. ****p < 0.0001. One-way ANOVA followed by the Dunnett’s post hoc test.

Article Snippet: Chemicals and Reagents: Antibodies specific for Phospho-STAT3Tyr705 (9145; 1:2000 for WB, 1:100 for immunofluorescence, 1:200 for immunohistochemistry), and STAT3 (4904; 1:2000 for WB) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Immunohistochemistry, Staining, Marker, Protein-Protein interactions, Concentration Assay, Inhibition

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization.

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Figure 5. Isoquercitrin inhibits STAT3 to improve endothelial and renal tubular epithelial cell injury. A–E) RT-qPCR detection of mRNA expression levels of interleukin (IL)-6, IL-1𝛽, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), and tumor growth factor beta (TGF-𝛽) in kidneys (n = 4). F) Immunofluorescence detection of endothelial cell injury marker ET-1 in kidneys (n = 6). G) WB detection of Kim-1 expression, a marker of renal tubular epithelial cell injury, in mouse kidneys (n = 4). RT-qPCR detection of lipocalin-2 (Lcn-2) (H) and tissue inhibitor of metalloproteinase 1 (TIMP-1) (I) mRNA expression levels in kidneys (n = 4). J) Immunohistochemistry and immunofluorescence detection of TNF-𝛼, p- STAT3, 𝛼-SMA, E-cadherin, vimentin, and aquaporin 2 (AQP2) expression in kidney tubules (n = 6). Electron microscopy observation of the morphology of mitochondria in renal tubular epithelial cells of mice (J) (n = 3). Data are presented as the mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by the Dunnett’s post hoc test.

Article Snippet: Chemicals and Reagents: Antibodies specific for Phospho-STAT3Tyr705 (9145; 1:2000 for WB, 1:100 for immunofluorescence, 1:200 for immunohistochemistry), and STAT3 (4904; 1:2000 for WB) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Expressing, Marker, Immunohistochemistry, Electron Microscopy

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization.

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Figure 6. Isoquercitrin alleviates high glucose or IL-6-induced endothelial cell injury by inhibiting STAT3 activity. RPMI Medium 1640 containing 45 mM glucose was used as a high glucose (HG) culture medium to induce human umbilical vein endothelial cells (HUVECs). RPMI Medium 1640 with a glucose concentration of 11 mM was used to culture HUVECs in the normal glucose (NG) control group. Subsequently, isoquercitrin (ISO) at concentrations of 5, 10, and 20 μM was added to the culture medium for intervention. A) WB detection of the p-STAT3, STAT3, and TGF-𝛽expression levels in HG-

Article Snippet: Chemicals and Reagents: Antibodies specific for Phospho-STAT3Tyr705 (9145; 1:2000 for WB, 1:100 for immunofluorescence, 1:200 for immunohistochemistry), and STAT3 (4904; 1:2000 for WB) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activity Assay, Concentration Assay, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization.

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Figure 7. Isoquercitrin inhibits STAT3 in renal tubular epithelial cells, reducing IL-6-induced pro-inflammatory and profibrotic cytokines. Human kidney 2 cells (HK2) cells were stimulated with 25 ng mL−1 IL-6, whereas HK2 cells without IL-6 stimulation were the normal control (NC) group. Subsequently, interventions were conducted with 5, 10, and 20 μM of isoquercitrin. A) WB detection of p-STAT3 and STAT3 expression in HK2 cells (n = 4). RT-qPCR detection of the mRNA expression of pro-inflammatory cytokines IL-6 (B), IL-1𝛽(C), and TNF-𝛼(D), macrophage chemoattractant protein MCP-1 (E), and profibrotic cytokine TGF-𝛽(F) (n = 4). G–J) Isoquercitrin inhibits EMT in renal tubular epithelial cells. Isoquercitrin downregulates the abnormally high expression of mesenchymal markers vimentin and 𝛼-SMA, while restoring the expression of the epithelial marker E-cadherin (n = 6). Data are presented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. One-way ANOVA followed by the Dunnett’s post hoc test.

Article Snippet: Chemicals and Reagents: Antibodies specific for Phospho-STAT3Tyr705 (9145; 1:2000 for WB, 1:100 for immunofluorescence, 1:200 for immunohistochemistry), and STAT3 (4904; 1:2000 for WB) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Control, Expressing, Quantitative RT-PCR, Marker

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Isoquercitrin Alleviates Diabetic Nephropathy by Inhibiting STAT3 Phosphorylation and Dimerization.

doi: 10.1002/advs.202414587

Figure Lengend Snippet: Figure 9. Schematic diagram showing the mechanism of isoquercitrin inhibiting STAT3 to ameliorate diabetic nephropathy. Isoquercitrin alleviates the injury of endothelial and renal tubular epithelial cells in diabetic nephropathy by inhibiting STAT3 phosphorylation and dimerization. Based on these actions, isoquercitrin reduces the expression of pro-inflammatory and profibrotic cytokines such as IL-1𝛽, IL-6, TNF-𝛼, ICAM-1, MCP-1, and TGF-𝛽, thereby alleviating renal inflammation and extracellular matrix accumulation.

Article Snippet: Chemicals and Reagents: Antibodies specific for Phospho-STAT3Tyr705 (9145; 1:2000 for WB, 1:100 for immunofluorescence, 1:200 for immunohistochemistry), and STAT3 (4904; 1:2000 for WB) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Expressing

Journal:

Article Title: HDAC4 inhibits cell cycle progression and protects neurons from cell death

doi: 10.1002/dneu.20637

Figure Lengend Snippet: (A and B) 5 day old cultures of CGNs were transfected with HDAC4 truncation mutants or GFP plasmids and switched to HK or LK medium for 24 hrs. Transfected neurons were identified by GFP fluorescence or Flag immunoreactivity and the status of the nuclei assessed by DAPI staining. Panel A demonstrates survival of cells overexpressing HDAC4 truncation mutants and GFP in LK medium as percent of control (Survival of GFP-transfected neurons in HK were set to 100%). The asterisks correspond to constructs that demonstrate statistically significant protection in LK when compared with control (survival of GFP transfected neurons in LK) (P<0.05). (B) Subcellular localization of HDAC4435 and HDAC4215 in HK and LK medium. Shown in red is Texas Red conjugated secondary antibody that binds the Flag tag present on the c-terminus of HDAC4435 and HDAC4215. Cell nuclei are stained using DAPI (blue).

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechonology (Santa Cruz, CA): Actin, HA, GFP, α-tubulin, c-Jun, Flag (rabbit origin), CDK5 and CDK1.

Techniques: Transfection, Fluorescence, Staining, Construct, FLAG-tag

Journal:

Article Title: HDAC4 inhibits cell cycle progression and protects neurons from cell death

doi: 10.1002/dneu.20637

Figure Lengend Snippet: (A) 5 days old CGNs were infected with Ad-HDAC4, Ad-GFP or left uninfected. 24 hrs after infection media was switched to HK or LK medium. Cell lysates were prepared 3 hrs after the treatments and subjected to Western blotting using antibodies against HDAC4, c-jun, p-JNK, pMEK, total MEK, pERK, total EREK, pGSK3β, MEF2D, pAkt, total Akt and α-tubulin (as loading control). “Input” shows an abundant protein in the Ponceau-S stained membrane. (B) 5 days old CGNs were transfected with HDAC4-N (Flag tag) or GFP plasmids 24 hrs before treatments with HK, LK or LK plus inhibitors. After 24 hrs of treatment, cells were subjected to immunocytochemistry using Flag antibody followed by DAPI staining. Following concentrations were used for each inhibitor: Gleevec (50 uM); LY294002 (10 uM); TSA (1 uM); U0126 (10 uM); KN62 (50 uM) and ML9 (20 uM). The graph demonstrates cell survival as percent of control (viability of GFP-transfected neurons in HK).

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechonology (Santa Cruz, CA): Actin, HA, GFP, α-tubulin, c-Jun, Flag (rabbit origin), CDK5 and CDK1.

Techniques: Infection, Western Blot, Staining, Transfection, FLAG-tag, Immunocytochemistry

Journal:

Article Title: HDAC4 inhibits cell cycle progression and protects neurons from cell death

doi: 10.1002/dneu.20637

Figure Lengend Snippet: (A) HEK293T cells were transfected with GFP, CDK1 and/or HDAC4 plasmids. Protein expression was allowed to persist for 22 hrs after which BrdU was added for an additional 2 hrs. Cells were then harvested and stained for HDAC4, GFP and BrdU. The graph demonstrates percentage of cells expressing protein of interest that are BrdU positive (the data has been normalized to % of GFP expressing BrdU positive cells) (P < 0.003). (B) CDK1 was immunoprecipitated from HEK293T cultures transfected with HDAC4, HDAC4-N or GFP plasmids and an in vitro kinase assay was performed in the presences of [γ-32P] ATP and Histone H1 as substrate. The extent of Histone H1 phosphorylation was detected by autoradiography. The panel showing Histone H1 is taken from staining of the membrane with Ponceau-S to demonstrate the similarity in the amount of substrate used. The membrane was also probed with CDK1antibody to show that similar amounts of kinase were pulled down. (C) CDK1 and CDK5 were immunoprecipitated from whole brain lysates obtained from mice lacking HDAC4 and wild-type littermates at P1. Kinase assays were performed as described in part B.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechonology (Santa Cruz, CA): Actin, HA, GFP, α-tubulin, c-Jun, Flag (rabbit origin), CDK5 and CDK1.

Techniques: Transfection, Expressing, Staining, Immunoprecipitation, In Vitro, Kinase Assay, Autoradiography

Journal: Life Science Alliance

Article Title: Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation

doi: 10.26508/lsa.202503563

Figure Lengend Snippet: (A) Schematic of domain structure and membrane topology of the CTLA4-FRB*-HA (CTLA4-FH) construct. (B) Representative Western blot (n = 3) of samples from non-induced FF-USP15 WT cells treated with cycloheximide (CHX, 100 μg/ml), TAK-243 (TAK, 1 μM), concanamycin (CMA, 100 nM), or epoxomicin (Epo, 1 μM) for indicated times before lysis. (C) Representative Western blot of HEK293 Flp-In T-REx FLAG-FKBP-USP15 WT (FF-USP15 WT) and C269S (FF-USP15 CS) cells, transfected with CTLA4-FH and treated with or without doxycycline (Dox; 0.1 μg/ml) for 18 h, then treated ± A/C (500 nM) for 24 h before lysis (see ). (D) Quantification of data represented in (C). Error bars represent SD from four (WT) or three (CS) independent colour-coded experiments. Statistical significance was determined using a two-way ANOVA with uncorrected Fisher’s LSD. **** P < 0.0001. (E, F) Representative Western blots (n = 2) of co-immunoprecipitation experiments to assess ternary complex formation upon A/C treatment. (E, F) FF-USP15 WT and CS cells were transfected, induced with Dox and treated ± A/C as in (C) before lysis and immunoprecipitation (IP) with anti-HA (E) or anti-FLAG (F) magnetic beads. ECD, extracellular domain; TM, transmembrane domain; CD, cytoplasmic domain; FRB*, FRB-T2098L; IB, immunoblot. Source data are available for this figure.

Article Snippet: Antibodies and other reagents used were as follows: anti-HA (MMS-101P; WB 1:1,000; Covance), anti-HA (NB600-362; WB 1:10,000; IF 1:250; Novus Biologicals), anti-HA (3724; WB 1:1,000; Cell Signaling Technology), anti-Actin (66009; WB 1:5,000; Proteintech), anti-HiBiT (N720A; WB 1:1,000; IF 1:1,000; Promega), anti-GAPDH (2118S; WB 1:1,000; Cell Signaling Technologies), anti-USP15 (A300-923A; WB 1:1,000; Bethyl Laboratories), anti-FLAG (F3165 or F1804; WB 1:1,000; IF 1:1,000; M2 Sigma-Aldrich), anti-DYKDDDDK (FLAG) Tag (D6W5B) (14793; IF 1:1,000; Cell Signaling Technology), anti-ubiquitin FK2 (PW8810; WB 1:500; Enzo), anti-USP8 antibody (AF7735; WB 1:500; R&D Systems), Wheat Germ Agglutinin (WGA) Alexa Fluor 647 (gift from Shankar Varadarajan, Liverpool), A/C Heterodimeriser (635055, 500 nM unless otherwise indicated; Takara/Clontech), doxycycline (D9891, 0.1 μg/ml; Sigma-Aldrich), cycloheximide (C7698, 100 μg/ml; Sigma-Aldrich), concanamycin A (C9705, 100 nM; Sigma-Aldrich), epoxomicin (324800, 1 μM; Merck Millipore), TAK-243 (S8341, 1 μM; Selleckchem), and Poly-L-lysine (P4832, solution, 0.01%; Merck Millipore).

Techniques: Membrane, Construct, Western Blot, Lysis, Transfection, Immunoprecipitation, Magnetic Beads

Journal: Life Science Alliance

Article Title: Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation

doi: 10.26508/lsa.202503563

Figure Lengend Snippet: (A) Cartoon of second generation Dox-inducible FF-USP15 WT and C269S (CS) HEK293 Flp-In T-REx cells constitutively expressing HiBiT-CTLA4-FH. (B) Treatment schedule and representative Western blot of FF-USP15 and CS cell pools stably expressing HiBiT-CTLA4-FH treated with or without doxycycline (Dox; 0.1 μg/ml) for 6 h, then treated for 18 h ± A/C (500 nM) before lysis. Par: parental uninduced (-Dox) FF-USP15 WT cells. (C, D) Quantification of data represented in (B). Error bars show SD from three independent colour-coded experiments. Statistical significance was determined using two-way ANOVA with uncorrected Fisher’s LSD. ** P < 0.01. (E) Representative immunofluorescence microscopy images of FF-USP15 and CS cell pools stably expressing HiBiT-CTLA4-FH, treated with doxycycline (Dox; 0.1 μg/ml) for 6 h, then treated for 24 h with A/C (500 nM) before fixation with methanol and staining with anti-HA (Alexa Fluor 488, green) and anti-FLAG (Alexa Fluor 647, magenta). Nuclear counterstain (DAPI) is shown in blue in the merged image. Images were acquired using a LSM800 confocal microscope (63x Oil objective). Scale bar: 15 μm. Yellow asterisks indicate cells that only express FF-USP15/CS but not CTLA4-FH. (F) Enlarged detail of boxed area shown in (E). Scale bar: 15 μm. FRT, Flp Recombination Target; IB, immunoblot. Source data are available for this figure.

Article Snippet: Antibodies and other reagents used were as follows: anti-HA (MMS-101P; WB 1:1,000; Covance), anti-HA (NB600-362; WB 1:10,000; IF 1:250; Novus Biologicals), anti-HA (3724; WB 1:1,000; Cell Signaling Technology), anti-Actin (66009; WB 1:5,000; Proteintech), anti-HiBiT (N720A; WB 1:1,000; IF 1:1,000; Promega), anti-GAPDH (2118S; WB 1:1,000; Cell Signaling Technologies), anti-USP15 (A300-923A; WB 1:1,000; Bethyl Laboratories), anti-FLAG (F3165 or F1804; WB 1:1,000; IF 1:1,000; M2 Sigma-Aldrich), anti-DYKDDDDK (FLAG) Tag (D6W5B) (14793; IF 1:1,000; Cell Signaling Technology), anti-ubiquitin FK2 (PW8810; WB 1:500; Enzo), anti-USP8 antibody (AF7735; WB 1:500; R&D Systems), Wheat Germ Agglutinin (WGA) Alexa Fluor 647 (gift from Shankar Varadarajan, Liverpool), A/C Heterodimeriser (635055, 500 nM unless otherwise indicated; Takara/Clontech), doxycycline (D9891, 0.1 μg/ml; Sigma-Aldrich), cycloheximide (C7698, 100 μg/ml; Sigma-Aldrich), concanamycin A (C9705, 100 nM; Sigma-Aldrich), epoxomicin (324800, 1 μM; Merck Millipore), TAK-243 (S8341, 1 μM; Selleckchem), and Poly-L-lysine (P4832, solution, 0.01%; Merck Millipore).

Techniques: Expressing, Western Blot, Stable Transfection, Lysis, Immunofluorescence, Microscopy, Staining

Journal: Life Science Alliance

Article Title: Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation

doi: 10.26508/lsa.202503563

Figure Lengend Snippet: (A) Half-life and degradation pathway assessment for stably expressed HiBiT-CTLA4-FH in uninduced FF-USP15 WT cells treated with cycloheximide (CHX, 100 μg/ml), TAK-243 (TAK, 1 μM), concanamycin (CMA, 100 nM), or epoxomicin (Epo, 1 μM) for indicated times before lysis. (B) Quantification of data shown in (A); bar chart shows all 6 h treatments. Error bars show range for two independent experiments. (C) Representative immunofluorescence microscopy images of FF-USP15 and CS cell pools stably expressing HiBiT-CTLA4-FH, treated with doxycycline (Dox; 0.1 μg/ml) for 6 h, then treated for 18 h ± A/C (500 nM). Cells were first stained with anti-HiBiT (Alexa Fluor 488, green) to label cell surface exposed HiBiT-CTLA4-FH, then fixed with PFA, permeabilised and counter-stained with anti-FLAG (Alexa Fluor 647, magenta). Nuclear counterstain (DAPI) is shown in blue in the merged image. Images were acquired on a LSM900, 63x oil objective, 2 × 2 tiles. Scale bar 20 μm. (D) Representative Western blot of FF-USP15 and CS cell pools stably expressing HiBiT-CTLA4-FH, treated with doxycycline (Dox; 0.1 μg/ml) for 6 h, then supplemented for 18 h ± A/C (500 nM) ± concanamycin (CMA, 100 nM) before lysis. (E) Quantification of data shown in (D). Error bars show range from two independent colour-coded experiments. (F) Representative immunofluorescence microscopy images FF-USP15 and CS cell pools stably expressing HiBiT-CTLA4-FH treated for 6 h with doxycycline (Dox; 0.1 μg/ml), then treated for 24 h ± A/C (500 nM) and supplemented ± concanamycin (CMA) for the last 6 h were fixed with methanol and stained with anti-HA (Alexa Fluor 488, green) and anti-FLAG (Alexa Fluor 647, magenta). Images were acquired using an LSM800 confocal microscope (63x Oil Objective). Scale bar: 15 μm. Source data are available for this figure.

Article Snippet: Antibodies and other reagents used were as follows: anti-HA (MMS-101P; WB 1:1,000; Covance), anti-HA (NB600-362; WB 1:10,000; IF 1:250; Novus Biologicals), anti-HA (3724; WB 1:1,000; Cell Signaling Technology), anti-Actin (66009; WB 1:5,000; Proteintech), anti-HiBiT (N720A; WB 1:1,000; IF 1:1,000; Promega), anti-GAPDH (2118S; WB 1:1,000; Cell Signaling Technologies), anti-USP15 (A300-923A; WB 1:1,000; Bethyl Laboratories), anti-FLAG (F3165 or F1804; WB 1:1,000; IF 1:1,000; M2 Sigma-Aldrich), anti-DYKDDDDK (FLAG) Tag (D6W5B) (14793; IF 1:1,000; Cell Signaling Technology), anti-ubiquitin FK2 (PW8810; WB 1:500; Enzo), anti-USP8 antibody (AF7735; WB 1:500; R&D Systems), Wheat Germ Agglutinin (WGA) Alexa Fluor 647 (gift from Shankar Varadarajan, Liverpool), A/C Heterodimeriser (635055, 500 nM unless otherwise indicated; Takara/Clontech), doxycycline (D9891, 0.1 μg/ml; Sigma-Aldrich), cycloheximide (C7698, 100 μg/ml; Sigma-Aldrich), concanamycin A (C9705, 100 nM; Sigma-Aldrich), epoxomicin (324800, 1 μM; Merck Millipore), TAK-243 (S8341, 1 μM; Selleckchem), and Poly-L-lysine (P4832, solution, 0.01%; Merck Millipore).

Techniques: Stable Transfection, Lysis, Immunofluorescence, Microscopy, Expressing, Staining, Western Blot

Journal: Life Science Alliance

Article Title: Targeted recruitment of USP15 enhances CTLA4 surface levels and restricts its degradation

doi: 10.26508/lsa.202503563

Figure Lengend Snippet: (A, B) Extracellular (A) and lytic (B) HiBiT assay to assess cell surface and total CTLA4 levels, respectively, performed on FF-USP15 and CS cells stably expressing HiBiT-CTLA4-FH treated ± doxycycline (Dox; 0.1 μg/ml) for 6 h, then treated for 18 h ± A/C (500 nM). Error bars show SD from three independent, colour-coded experiments each performed as technical triplicates. Statistical significance was determined using two-way ANOVA with uncorrected Fisher’s LSD. **** P < 0.0001 and ** P < 0.01. (C) FF-USP15 and CS cells stably expressing HiBiT-CTLA4-FH were transfected with either USP8- or non-targeting (NT1) siRNA for 48 h, then treated for 6 h with doxycycline (Dox; 0.1 μg/ml), and supplemented for another 20 h ± A/C (500 nM). Cells were lysed and samples subjected to a pulldown using TUBEs to enrich ubiquitylated proteins, followed by Western blotting with anti-HA. Representative Western blot depicting the Ub-HiBiT-CTLA4-FH signal. (D) Treatment schedule and quantification of data represented in (C). Shown is the Ub-HiBiT-CTLA4-FH enriched in the TUBE pulldown normalised to HiBiT-CTLA4-FH in the input samples. Error bars show SD from three independent colour-coded experiments. Statistical significance was determined using two-way ANOVA with uncorrected Fisher’s LSD. **** P < 0.0001. (E) USP15 RapTag in action. Left panel: At steady state, cell surface CTLA4 is rapidly internalised and sorted in a ubiquitin-dependent manner for degradation in the lysosome. Middle panel: A/C-induced recruitment of active USP15 deubiquitylates CTLA4 and thereby opposes its internalisation whereas also promoting recycling, resulting in a net increase in cell surface CTLA4. Right panel: Inactive USP15 fails to deubiquitylate CTLA4 and is itself destabilised as a bystander by sorting with CTLA4 to the lysosome. IB, immunoblot; CTRL, control beads. Source data are available for this figure.

Article Snippet: Antibodies and other reagents used were as follows: anti-HA (MMS-101P; WB 1:1,000; Covance), anti-HA (NB600-362; WB 1:10,000; IF 1:250; Novus Biologicals), anti-HA (3724; WB 1:1,000; Cell Signaling Technology), anti-Actin (66009; WB 1:5,000; Proteintech), anti-HiBiT (N720A; WB 1:1,000; IF 1:1,000; Promega), anti-GAPDH (2118S; WB 1:1,000; Cell Signaling Technologies), anti-USP15 (A300-923A; WB 1:1,000; Bethyl Laboratories), anti-FLAG (F3165 or F1804; WB 1:1,000; IF 1:1,000; M2 Sigma-Aldrich), anti-DYKDDDDK (FLAG) Tag (D6W5B) (14793; IF 1:1,000; Cell Signaling Technology), anti-ubiquitin FK2 (PW8810; WB 1:500; Enzo), anti-USP8 antibody (AF7735; WB 1:500; R&D Systems), Wheat Germ Agglutinin (WGA) Alexa Fluor 647 (gift from Shankar Varadarajan, Liverpool), A/C Heterodimeriser (635055, 500 nM unless otherwise indicated; Takara/Clontech), doxycycline (D9891, 0.1 μg/ml; Sigma-Aldrich), cycloheximide (C7698, 100 μg/ml; Sigma-Aldrich), concanamycin A (C9705, 100 nM; Sigma-Aldrich), epoxomicin (324800, 1 μM; Merck Millipore), TAK-243 (S8341, 1 μM; Selleckchem), and Poly-L-lysine (P4832, solution, 0.01%; Merck Millipore).

Techniques: Stable Transfection, Expressing, Transfection, Western Blot, Ubiquitin Proteomics, Control