Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 1. FUS is in a complex with MITF. A, Coomassie-stained FLAG-immunoprecipitated samples run on an 8.5% SDS-polyacrylamide gel; lane 1 (mock), FLAG-immunoprecipitated lysates from parental RAW264.7 C4 cell line; lane 2 (FM), FLAG immunoprecipitated lysates from RAW264.7 C4 cell line stably expressing FLAG-MITF. Lane 3 (M) marker, FUS and MITF based on size are indicated on the right. B, pulldown of RAW264.7 C4 cell lysates using GSH-Sepharose beads incubated either with GST (lane 1) or GST-MITF (lane 2) and probed with anti-FUS antibody. Input controls for RAW264.7 C4 lysates and GST beads are depicted in middle and bottom panels, respectively. C, top panel, schematic diagram of full-length and N- and C-terminal FUS constructs used for MITF interaction mapping. Ser-, Tyr-, Gly-, and Gln-rich N-terminal activation domain (AD) and C-terminal RGG-rich RNA binding domains (RBD) are shown. Bottom panel, coimmunoprecipitation studies in COS-7 cell lysates using full-length FLAG-MITF (lanes 2–4), full-length V5-FUS (lane 1 and 2), V5-tagged N-terminal FUS (lane 3, N; aa 1–259) or V5-tagged C-terminal FUS (lane 4, C; aa 260–518) immunoprecipitated (IP) with anti-FLAG antibody and probed with anti-V5 antibody. Input controls for V5-FUS and FLAG-MITF are shown. D, top panel, schematic diagram of full-length and N- and C-terminal MITF constructs used for FUS interactionmapping.N-terminalactivationdomains(AD1andAD2denotedasAD),basic(B),helix-loop-helix(HLH),andleucinezipper(LZ)domainsareshown. Ser-73 ERK phosphorylation site and Ser-307 p38 MAPK phosphorylation site as well as Lys-182 and Lys-316 sumoylation sites are also denoted. Bottom panel, immunoprecipitates pulled with FLAG antibody from COS-7 cell lysates expressing full-length FLAG-MITF (lane 1 and 3), full-length V5-FUS (lanes 2–5), FLAG-tagged N-terminal (aa 1–178) and C-terminal (aa 178–419) MITF (lanes 4 and 5, respectively) and immunoblotted (IB) with anti-V5 antibody. Loading controls for V5-FUS and FLAG-MITF are shown at the bottom.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Staining, Immunoprecipitation, Stable Transfection, Expressing, Marker, Incubation, Construct, Activation Assay, RNA Binding Assay, Phospho-proteomics

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 2. FUS-MITF complex formation is mediated by CSF-1/RANKL signaling. A, immunoprecipitation (IP) of endogenous MITF from murine bone marrow- derivedmyeloidcellstreatedwithCSF-1alone(lane1)orCSF-1andRANKLfor24and72h(lanes2and3,respectively)andimmunoblotted(IB)withFUSantibody(top panel).InputcontrolsforFUSandMITFforthesametreatmentsareshowninthemiddleandbottompanels,respectively.B,immunoprecipitationofendogenousFUS and BRG1 using anti-FLAG antibody in RAW264.7 C4 cells stably expressing FLAG-MITF in response to CSF-1 and RANKL treatments. Lane 1, cells treated with CSF-1 alone; lanes 2–5, cells treated with CSF-1 and RANKL for 6, 24, 48, and 72 h, respectively. Top two panels show the immunoblotting with FUS- and BRG1-specific antibodies on immunoprecipitates. Loading controls for FUS, BRG1, and FLAG-MITF are shown in the bottom three panels. C, RAW264.7 C4 cells were transiently transfectedwithFLAG-taggedwildtype(wt,leftpanel)orS307AformofMITF(rightpanel),andtheinteractionofFUSwasexaminedwithrespecttoCSF-1andRANKL treatmentovertheindicatedtimecourse(top)byimmunoprecipitationwithFLAGantibodyfollowedbyimmunoblottingwithFUSantibody.Lane1,transfectedcells as above treated with CSF-1 alone;lanes 2–4,transfected cells treated with CSF-1 and RANKL for 1, 8, and 16 h, respectively. Input controls for FUS and FLAG-MITF are depictedinlowertwopanels.D,lysatesfromCOS-7cellsoverexpressingFLAG-MITF(lanes2–4),V5-FUS(lanes1–4),andeitherconstitutivelyactiveMYC-RAF(lane3)or MKK6 (lane 4) were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted with anti-V5 antibody (top panel). Whole cell lysates were also immunoblotted with anti-V5, anti-MYC, anti-pp38, or anti-FLAG antibodies (input controls, bottom four panels).

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Stable Transfection, Expressing, Western Blot, Transfection

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 3. FUS is a coactivator of MITF in CSF-1/RANKL-mediated induc- tion of MITF target genes. A, soluble chromatin from BMMs treated either with CSF-1 alone or CSF-1 and RANKL for the indicated time points were immunoprecipitated with FUS antibody, and enrichment of Acp5 and Ctsk promoters was analyzed by qPCR. The top panel shows the relative positions of the oligonucleotides used with respect to the TSS of each gene. B, evalua- tion of FUS knockdown by shRNA FUS constructs. Left panel, either scrambled or shFUS retroviral constructs were nucleofected into RAW264.7 C4 F-M cells, and24hpost-infection,celllysateswereprobedforFUSproteinandTUBULIN protein (loading control). Right panel, mRNA levels of Fus in presence and absence of CSF-1 and RANKL 24 h post-infection with either scrambled (Scr) or shRNA FUS by qRT-PCR. C, RAW264.7 C4 F-M cells infected and treated as in B were analyzed for mRNA expression of Acp5 and Ctsk in the presence or absenceofCSF-1andRANKLbyqRT-PCR.Allexperimentsweretheaverageof at least two independent biological sets done in duplicate, and the error bars indicate S.D.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Knockdown, shRNA, Construct, Retroviral, Infection, Control, Quantitative RT-PCR, Expressing

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 4. FUS, MITF, and BRG1 complex formation is mediated by Ser(P)-307 of MITF. A, coimmunoprecipitation of V5-FUS in COS-7 cells overexpressing either BRG1 and FLAG-MITF (lane 1), BRG1 and V5-FUS (lane 2), or all three constructs (lane 3) and immunoblotted (IB) with anti-BRG1 antibody (top panel) or anti-FLAG antibody (2nd panel from top). Input lysates for V5, BRG1, and FLAG-MITF are denoted in subsequent panels. B, coimmunoprecipitation of FLAG- tagged wild type MITF (lanes 2 and 4) or S307A mutant MITF (lanes 3 and 5) in the absence (lanes 1–3) or presence (lanes 4 and 5) of BRG1. Top panel shows immunoblot of V5 on FLAG immunoprecipitates (IP), and subsequent panels depict input controls. C, relative quantification of the band areas in B (top panel) using ImageJ software. D, relative mRNA expression of Acp5, Ctsk, and Fus in RAW264.7 C4 cells that were nucleofected with either plasmid encoding V5-Fus alone, Brg1 alone, or both plasmids. Post-nucleofection the cells were incubated in the presence (dark bars) or absence (light bars) of CSF-1 and RANKL for 24 h. All experiments were performed on at least two biological sets done in duplicate, and the error bars indicate S.D. E, soluble chromatin from RAW264.7 C4 cells nucleofected with either scrambled (Scr) or shFUS retroviral constructs and treated with CSF-1 and RANKL for 3 days were immunoprecipitated with either FUS antibody (left panel) or BRG1 antibody (right panel), and enrichment of the Ctsk promoter was analyzed by qPCR. All experiments were performed on at least two biological sets done in duplicate, and the error bars indicate S.D.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Construct, Mutagenesis, Western Blot, Quantitative Proteomics, Software, Expressing, Plasmid Preparation, Incubation, Retroviral, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 5. MITF phospho-sumoylation switch is regulated by CSF-1 and RANKL signaling. A, cell extracts prepared under denaturing lysis conditions from primary BMMs treated with either CSF1 (lane 1) or CSF1/RANKL (lane 2) for 72 h or COS-7 cells overexpressing FLAG-MITF and HA-SUMO (lane 3) were immunoblotted with anti-MITF antibody (top panel). GAPDH loading control is shown in the bottom panel. B, GSH-Sepharose pulldowns from RAW264.7 C4 cell lysates overexpressing GST-MITF and HA-SUMO1 treated with CSF-1 and RANKL for times (in hours) indicated below were immunoblotted (IB) with SUMO1 antibody and GST antibody (1st and 2nd panels from left). Input MITF and HA are denoted on right two panels. C, GST pulldown using COS-7 cells overexpressing GST-MITFalone(lane1),GST-MITFandHA-SUMO1(lane2),orGST-MITFandHA-SUMO1withconstitutivelyactiveMYC-RAForMKK6(lanes3and4,respectively) were analyzed by immunoblotting with GST antibody (top panel) or SUMO1 antibody (2nd from top). Inputs probed for phosphorylated p38 MAPK and MYC are shown in lower two panels.

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Lysis, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Multifunctional Protein Fused in Sarcoma (FUS) Is a Coactivator of Microphthalmia-associated Transcription Factor (MITF)

doi: 10.1074/jbc.m113.493874

Figure Lengend Snippet: FIGURE 6. Sumoylation of MITF at Lys-316 inhibits interaction of FUS with MITF. A, cell extracts from either FLAG-MITF (wt) (lanes 1, 2, and 6) or FLAG-MITF with point or double mutations (K182R (lane 3), K316R (lane 4), K182R/K316R (K182/316R; lane 5), HA-SUMO1 (lanes 2–6), and MKK6 (lane 6)) expressing COS-7 cells were immunoprecipitated (IP) with anti-FLAG antibody (IP: FLAG). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted (IB) with anti-HA antibody. Whole cell lysates were also immunoblotted with anti-FLAG, anti-HA, or anti-Pp38 antibody (input controls). B, cell extracts from either FLAG-MITF (wt) (lanes 2 and 7), FLAG-MITF K182R (K182R; lanes 3 and 8), FLAG-MITF K316R (K316R; lanes 4 and 9), FLAG-MITF K182R/K316R (K182/316R; lanes 5 and 10), V5-FUS (lanes 1–10), and HA-SUMO1 (lanes 6–10)-expressing COS-7 cells were immunoprecipitated with anti-FLAG antibody (IP: FLAG). Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-V5 or anti-FLAG antibody. Whole cell lysates were also immunoblotted with anti-V5 antibody (lower panel). C, lysates from COS-7 cells expressing FLAG-MITF (wt) (lane 2 and 4) or K316R FLAG-MITF mutant (lane 3 and 5) and V5-FUS (lanes 1–5) and HA-SUMO1 (lane 4 and 5) were immunoprecipitated with anti-FLAG antibody (IP: FLAG) and immunoblotted anti-BRG1 antibody (top panel). Whole cell lysates were also immunoblotted with anti-FLAG, anti-V5, or anti-BRG1 antibodies (input controls, bottom three panels).

Article Snippet: Antibodies used were as follows: FLAG (mouse monoclonal, M2; Santa Cruz Biotechnology); V5 (mouse monoclonal, Invitrogen); FUS (rabbit polyclonal, Bethyl Laboratories, Inc.); GST (mouse monoclonal, Sigma); MITF and MITF PS307 (rabbit polyclonal affinity purified (5)); MITF (mouse monoclonal, Abcam), c-MYC (mouse monoclonal, Santa Cruz Biotechnology); HA (mouse monoclonal, Sigma); BRG1 (rabbit polyclonal affinity purified (24)); SUMO1 (mouse monoclonal, Santa Cruz Biotechnology); pp38 (rabbit polyclonal, Cell Signaling), and GAPDH (rabbit polyclonal, Santa Cruz Biotechnology).

Techniques: Expressing, Immunoprecipitation, SDS Page, Mutagenesis

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: Chalcone 21-21 inhibits the expression of melanogenesis-related proteins. ( A , B ) B16F10 cells were incubated for 24 h with vehicle (0.1% DMSO) alone or various concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). The levels of TRP1, TRP2, and MITF were determined by Western blot or RT-PCR analyses. GAPDH served as a loading control. ( C ) B16F10 cells were transiently transfected with a reporter construct of MITF together with the pRL-TK. The cells were incubated for 24 h with vehicle (0.1% DMSO) alone or with various concentrations of chalcone 21-21 in the absence or presence of α-MSH (10 nM). Luciferase activity was measured and firefly luciferase activities were normalized to that of Renilla luciferase. Results are represented as mean ±SD from three independent experiments ( n = 3). p -values of # p < 0.005 compared to the vehicle-treated group and * p < 0.05 and ** p < 0.005 compared to the α-MSH-treated group were considered statistically significant.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Transfection, Construct, Luciferase, Activity Assay

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: Primer sequences for the RT-PCR.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Sequencing

Journal: Molecules

Article Title: Evaluation and Optimization of the Anti-Melanogenic Activity of 1-(2-Cyclohexylmethoxy-6-hydroxy-phenyl)-3-(4-hydroxymethyl-phenyl)-propenone Derivatives

doi: 10.3390/molecules24071372

Figure Lengend Snippet: A schematic diagram illustrating the proposed action mechanisms of chalcone 21-21. Chalcone 21-21 specifically targets the ERK/CREB signaling, thereby inhibiting MITF and MITF-regulated target genes including those encoding tyrosinase, TRP1, and TRP2. AC, adenylate cyclase; α-MSH, alpha-melanocyte stimulating hormone; ATP, adenosine triphosphate; cAMP, cyclic adenosine monophosphate; CREB, cAMP response element-binding protein; ERK, extracellular signal-regulated kinase; MAP kinase, mitogen-activated protein kinase; MC1R, melanocortin receptor type 1; MITF, microphthalmia-associated transcription factor; PKA, protein kinase A; ROS, reactive oxygen species; TRP, tyrosinase-related protein; TYR, tyrosinase.

Article Snippet: Primary antibodies specific for tyrosinase, TRP1, TRP2, and MITF were obtained from Novus Biologicals, and phospho-ERK1/2, ERK1/2, phospho-CREB, CREB, and GAPDH were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Binding Assay

Journal: STAR Protocols

Article Title: An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells

doi: 10.1016/j.xpro.2022.101803

Figure Lengend Snippet: Characterization of RPE (A–E) Immunostaining of iPSC derived RPE cultures for the marker expression during stages of retinal differentiation. Images showing the expression of RPE commitment marker MITF (A), melanocyte protein- PMEL17 (B), RPE maturation markers TYRP1, RPE65 and TYR (C-E)and tight junction marker ZO-1 (D and E). (F) Flow cytometry analysis of iPSC derived RPE cells for positive (MITF, RPE65, TYRP1) and negative (OCT4) markers. (G and H) Gene expression analysis using qPCR for selected markers represented as fold change compared to iPSC. All the positive markers (G) have higher expression and the negative markers (H) show a negligible expression in iPSC derived RPE. (I) ELISA based quantification of secreted PEDF from the in vivo culture supernatant from apical, basal and 2D RPE cultures. Images and graphs are representative of minimum three independent experiments. Scale bars 100 μm.

Article Snippet: MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (1:150) , Novus Biologicals , MA5-32554.

Techniques: Immunostaining, Derivative Assay, Marker, Expressing, Flow Cytometry, Gene Expression, Enzyme-linked Immunosorbent Assay, In Vivo

Journal: STAR Protocols

Article Title: An improved protocol for generation and characterization of human-induced pluripotent stem cell-derived retinal pigment epithelium cells

doi: 10.1016/j.xpro.2022.101803

Figure Lengend Snippet:

Article Snippet: MiTF Recombinant Rabbit Monoclonal Antibody (JF100-01) (1:150) , Novus Biologicals , MA5-32554.

Techniques: Recombinant, Modification, Gentle, Saline, Knock-Out, Enzyme-linked Immunosorbent Assay, SYBR Green Assay, cDNA Synthesis, Sequencing, Generated, Software, Suspension, Real-time Polymerase Chain Reaction, Microscopy