Journal: International Journal of Molecular Medicine

Article Title: Altered expression of glycoprotein non-metastatic melanoma protein B in the distal sciatic nerve following injury

doi: 10.3892/ijmm.2020.4559

Figure Lengend Snippet: Expression and secretion of NCAM and N-cadherin in Schwann cells following treatment with rhGPNMB. (A) Reverse transcription-quantitative PCR (n=6), (B and C) western blot analysis (n=6) and (D and E) ELISA (n=8) were performed to detect the expression of NCAM and N-cadherin. * P<0.05 vs. 0 nM. # P<0.05 vs. 10 nM. $ P<0.05 vs. 50 nM. Analyses were performed using one-way analysis of variance and Tukey's post hoc test. NCAM, neural cell adhesion molecule; N-cadherin, N-cadherin; rhGPNMB, recombinant human glycoprotein non-metastatic melanoma protein B.

Article Snippet: ELISA kits was used to measure levels of NGF (cat. no. ER0143), BDNF (cat. no. ER0102), NT-3 (cat. no. ER0144; all Biotech Well Co., Ltd.), NCAM (cat. no. EK1562) and N-cadherin (cat. no. EK1156, both Wuhan Boster Biological Technology, Ltd.) in culture medium following the manufacturer's protocol.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant

Journal:

Article Title: FIBROBLAST GROWTH FACTOR 2 ENHANCES STRIATAL AND NIGRAL NEUROGENESIS IN THE ACUTE 1-METHYL-4-PHENYL-1,2,3, 6-TETRAHYDROPYRIDINE MODEL OF PARKINSON'S DISEASE

doi: 10.1016/j.neuroscience.2008.02.063

Figure Lengend Snippet: FGF-2 stimulates neurogenesis in the striatum and SN of MPTP-treated mice. Quantitation of BrdU-positive profiles in SVZ (A), DCX/BrdU-positive cells in SN (B), TUC-4/BrdU-positive cells in striatum (C), βIII-tubulin/BrdU-positive cells in striatum (D), and PSA-NCAM/BrdU-positive cells in SN (E). White bars, saline; black bars, MPTP. Data are mean±S.E., n=4. * P<0.01 compared with vehicle plus saline group; # P<0.05, compared with vehicle plus MPTP group (Newman-Keuls post hoc test).

Article Snippet: Fluorescence immunohistochemistry Sections were fixed with 4% paraformaldehyde in PBS for 1 h at room temperature, washed twice with PBS, and incubated in 2 M HCl at 37 °C for 1 h. After washing again, sections were incubated with blocking solution, then with primary antibodies at 4 °C overnight, and with secondary antibodies in blocking solution at room temperature for 2 h. The primary antibodies used were mouse monoclonal anti-BrdU (Roche, Indianapolis, IN, USA; 2 μ g/ml), sheep polyclonal anti-BrdU (Biodesign, Saco, ME, USA; 25 μ g/ml), rabbit anti-Ki-67 antigen (Zymed, South San Francisco, CA, USA; 1:100), mouse monoclonal anti-neuronal nuclear antigen (NeuN) (Chemicon; 1:200), affinity-purified goat polyclonal anti-doublecortin (DCX) (Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:200), rabbit polyclonal anti-TUC-4 (Chemicon; 1:1000), mouse monoclonal anti- β III-tubulin (Caltag Laboratories, Burlingame, CA, USA; 1:400), mouse monoclonal anti-glial fibrillary acidic protein (GFAP) (Sigma; 1:400), rat anti-mouse CD11b (Serotec Inc., Raleigh, NC, USA; 1:50), mouse monoclonal anti-2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPS) (Chemicon; 1:500), rat anti-polysialic acid-neural cell adhesion molecule (PSA-NCAM) (BD Biosciences, San Jose, CA, USA; 1:100), and rabbit polyclonal anti-tyrosine hydroxylase (TH) (Chemicon; 1:200).

Techniques: Quantitation Assay

Journal: Neuropathology

Article Title: Praja1 RING ‐finger E3 ubiquitin ligase is a common suppressor of neurodegenerative disease‐associated protein aggregation

doi: 10.1111/neup.12840

Figure Lengend Snippet: Suppressive effects of E3 ubiquitin ligases on neuronal TDP‐43 aggregate formation. (A) Schematic presentation of the experiments for adenoviral TDP‐43 aggregate formation. The differentiated 1464R cells were infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human wild‐type (WT; AxDsRhTDP43WTFL) and C‐terminal fragment (CTF; AxDsRhTDP43CTFFL) TDP‐43 and EGFP‐tagged human genes of interest (GOIs; AxhGOIEGFPs, i.e. AxhHSF1EGFP, AxhPJA1EGFP, AxhPJA1ΔREGFP, AxhPRKNEGFP, AxhRNF112EGFP or AxhRNF220EGFP), followed by incubation with 0.5 μ m MG‐132. (B) Western blot analysis of suppressive effects of adenoviral HSF1 and E3 ubiquitin ligases on phosphorylation and aggregate formation of adenoviral TDP‐43. (C) Densitometric analysis of the Western blot data of RIPA‐insoluble phosphorylated and total CTF TDP‐43 (arrowheads in B; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG‐132. Results are presented as mean ± SD. Statistical comparison was performed by a two‐tailed unpaired t ‐test (* P < 0.05). (D) The co‐immunoprecipitation (Co‐IP) assay showing that PJA1 (1), Parkin (2), RNF112 (3) and RNF220 (4) all bind to WT and CTF TDP‐43 that are ubiquitinated. (E) Fluorescence microscopy of TuJ1‐immunoreactive neurons infected with adenoviruses expressing DsRed‐tagged human WT and CTF TDP‐43 (AxhDsRTDP43 WT + CTF FL) and EGFP‐tagged human HSF1, PJA1, PJA1ΔR, Parkin (PRKN), RNF112 and RNF220 in the presence of MG‐132. The nucleus was counterstained with Hoechst 33342.

Article Snippet: The adult rat neural stem cell line 1464R that we have established (Cat. No. #T0746; Applied Biological Materials, Canada, Cat. No. #RNSCL‐C; Cosmo Bio, Tokyo, Japan) was cultured as previously described., , , To differentiate 1464R cells into neuronal and glial cells, dissociated 1464R cells were seeded on poly‐ l ‐lysine (PLL; #P1524; Sigma‐Aldrich, St Louis, MO, USA)‐coated 12‐well plates or 9‐mm ACLAR round coverslips (Allied Fibers & Plastics, Pottsville, PA, USA) at a density of 4–6 × 10 5 cells per well and 1–2 × 10 4 cells per coverslip, respectively, and maintained in differentiation medium composed of F12 medium (Cat. No. #11765‐054; Thermo Fisher Scientific, Waltham, CA, USA) containing 5% fetal bovine serum (Moregate, Bulimba, Australia), 0.5% N2 supplement (Cat. No. #17502‐048; Thermo Fisher), 1% B27 supplement (Cat. No. #17504‐044; Thermo Fisher), 1 μ m all‐trans retinoic acid (ATRA; Cat. No. # R2625; Sigma‐Aldrich), 50 units/mL penicillin and 50 μg/mL streptomycin in 5% CO 2 at 37°C.

Techniques: Ubiquitin Proteomics, Infection, Expressing, Incubation, Western Blot, Phospho-proteomics, Control, Comparison, Two Tailed Test, Co-Immunoprecipitation Assay, Fluorescence, Microscopy

Journal: Neuropathology

Article Title: Praja1 RING ‐finger E3 ubiquitin ligase is a common suppressor of neurodegenerative disease‐associated protein aggregation

doi: 10.1111/neup.12840

Figure Lengend Snippet: Suppression of the aggregate formation of neurodegenerative disease‐associated proteins by PJA1. (A) Schematic presentation of the experiments for adenovirus‐transduced protein aggregate formation. The 1464R‐derived neuronal cells were infected with adenoviruses expressing DsRed‐ and FLAG‐tagged genes of interest (GOI), i.e. human wild‐type (WT) and P525L FUS (B), WT and G93A SOD1 (C), WT and A53T α‐synuclein (D), ataxin‐3 Q28 and Q84 (E), and huntingtin exon 1 Q23 and Q74 (F), and EGFP‐tagged PJA1 or PJA1ΔR, followed by incubation with 0.5 μ m MG‐132. (B1–4) Suppression of FUS aggregate formation by PJA1. (B1) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human WT and P525L FUS (AxDsRhFUSWTFL, AxDsRhFUSP525LFL) and EGFP‐tagged PJA1 (AxhPJA1EGFP) or PJA1ΔR (AxhPJA1ΔREGFP) in the presence f MG‐132. (B2) Densitometric analysis of FLAG Western blot data of RIPA‐insoluble WT and P525L FUS versus PJA1 and PJA1ΔR (PJA1D) (arrowheads in B1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (* P < 0.05). (B3) The co‐immunoprecipitation (Co‐IP) assay showing that PJA1 and PJA1ΔR bind to WT and P525L FUS that are ubiquitinated (arrowheads). (B4) Fluorescence microscopy of TuJ1‐immunoreactive neurons infected with adenoviruses expressing DsRed‐tagged human P525L FUS and EGFP‐tagged human PJA1 in the presence of MG‐132. The nucleus was counterstained with Hoechst 33342. (C1–4) Suppression of SOD1 aggregate formation by PJA1. (C1) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human WT and G93A SOD1 (AxDsRhSOD1WTFL, AxDsRhSOD1G93AFL) and EGFP‐tagged PJA1 (AxhPJA1EGFP) or PJA1ΔR (AxhPJA1ΔREGFP) in the presence of MG‐132. (C2) Densitometric analysis of the FLAG Western blot data of RIPA‐insoluble WT and G93A SOD1 versus PJA1 and PJA1ΔR (PJA1D) (arrowheads in C1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (* P < 0.05). (C3) The Co‐IP assay showing that PJA1 and PJA1ΔR bind to WT and G93A SOD1 that are ubiquitinated (arrowheads). (C4) Fluorescence microscopy of TuJ1‐immunoreactive neurons infected with adenoviruses expressing DsRed‐tagged human G93A SOD1 and EGFP‐tagged human PJA1 in the presence of MG‐132. The nucleus was counterstained with Hoechst 33342. (D1–4) Suppression of α‐synuclein aggregate formation by PJA1. (D1) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human WT and A53T α‐synuclein (AxDsRhSNCAWTFL, AxDsRhSNCAA53TFL) and EGFP‐tagged PJA1 (AxhPJA1EGFP) or PJA1ΔR (AxhPJA1ΔREGFP) in the presence of MG‐132. (D2) Densitometric analysis of the FLAG and phosphorylated α‐synuclein Western blot data of RIPA‐insoluble WT and A53T α‐synuclein versus PJA1 and PJA1ΔR (PJA1D) (arrowheads in D1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (* P < 0.05). (D3) The Co‐IP assay showing that PJA1 and PJA1ΔR bind to WT and A53T α‐synuclein that are ubiquitinated (arrowheads). (D4) Fluorescence microscopy of TuJ1‐immunoreactive neurons infected with adenoviruses expressing DsRed‐tagged human A53T α‐synuclein and EGFP‐tagged human PJA1 in the presence of MG‐132. The nucleus was counterstained with Hoechst 33342. (E1–4) Suppression of ataxin‐3 aggregate formation by PJA1. (E1) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human ataxin‐3 Q28 and Q84 (AxDsRhATXN3Q28FL, AxDsRATXN3Q84FL) and EGFP‐tagged PJA1 (AxhPJA1EGFP) or PJA1ΔR (AxhPJA1ΔREGFP) in the presence of MG‐132. (E2) Densitometric analysis of the FLAG Western blot data of RIPA‐insoluble ataxin‐3 Q28 and Q84 versus PJA1 and PJA1ΔR (PJA1D) (arrowheads in E1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (* P < 0.05). (E3) The Co‐IP assay showing that PJA1 and PJA1ΔR bind to ataxin‐3 Q28 and Q84 that are ubiquitinated (arrowheads). (E4) Fluorescence microscopy of 1464R‐derived TuJ1‐immunoreactive neurons infected with adenoviruses expressing DsRed‐tagged human ataxin‐3 Q84 and EGFP‐tagged human PJA1 in the presence of MG‐132. The nucleus was counterstained with Hoechst 33342. (F1–4) Suppression of ataxin‐3 aggregate formation by PJA1. (F1) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged huntingtin exon 1/Q23 and Q74 (AxDsRhHttEx1Q23FL, AxDsRhHttEx1Q74FL) and EGFP‐tagged PJA1 (AxhPJA1EGFP) or PJA1ΔR (AxhPJA1ΔREGFP) in the absence or presence of MG‐132. (F2) Densitometric analysis of the FLAG Western blot data of RIPA‐insoluble huntingtin exon 1 Q23 and Q74 versus PJA1 and PJA1ΔR (PJA1D) (arrowheads in F1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (* P < 0.05). (E3) The Co‐IP assay showing that PJA1 and PJA1ΔR bind to huntingtin exon 1/Q23 and Q74 that are ubiquitinated (arrowheads). (E4) Fluorescence microscopy of 1464R‐derived TuJ1‐immunoreactive neurons infected with adenoviruses expressing DsRed‐tagged human huntingtin exon 1 Q74 and EGFP‐tagged human PJA1 in the presence of MG‐132. The nucleus was counterstained with Hoechst 33342.

Article Snippet: The adult rat neural stem cell line 1464R that we have established (Cat. No. #T0746; Applied Biological Materials, Canada, Cat. No. #RNSCL‐C; Cosmo Bio, Tokyo, Japan) was cultured as previously described., , , To differentiate 1464R cells into neuronal and glial cells, dissociated 1464R cells were seeded on poly‐ l ‐lysine (PLL; #P1524; Sigma‐Aldrich, St Louis, MO, USA)‐coated 12‐well plates or 9‐mm ACLAR round coverslips (Allied Fibers & Plastics, Pottsville, PA, USA) at a density of 4–6 × 10 5 cells per well and 1–2 × 10 4 cells per coverslip, respectively, and maintained in differentiation medium composed of F12 medium (Cat. No. #11765‐054; Thermo Fisher Scientific, Waltham, CA, USA) containing 5% fetal bovine serum (Moregate, Bulimba, Australia), 0.5% N2 supplement (Cat. No. #17502‐048; Thermo Fisher), 1% B27 supplement (Cat. No. #17504‐044; Thermo Fisher), 1 μ m all‐trans retinoic acid (ATRA; Cat. No. # R2625; Sigma‐Aldrich), 50 units/mL penicillin and 50 μg/mL streptomycin in 5% CO 2 at 37°C.

Techniques: Derivative Assay, Infection, Expressing, Incubation, Western Blot, Control, Co-Immunoprecipitation Assay, Fluorescence, Microscopy

Journal: Neuropathology

Article Title: Praja1 RING ‐finger E3 ubiquitin ligase is a common suppressor of neurodegenerative disease‐associated protein aggregation

doi: 10.1111/neup.12840

Figure Lengend Snippet: Suppression of the aggregate formation of neurodegenerative disease‐associated proteins by PJA1 in the presence of proteasome inhibitor lactacystin or bortezomib. FLAG Western blot analysis of 1464R‐derived neurons infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human wild‐type (WT) and P525L FUS, WT and G93A SOD1, WT and A53T α‐synuclein, and ataxin‐3 Q28 and G84 (arrowheads), and EGFP‐tagged PJA1 in the presence of 1 μ m lactacystin (A) or 0.5 μ m bortezomib (B).

Article Snippet: The adult rat neural stem cell line 1464R that we have established (Cat. No. #T0746; Applied Biological Materials, Canada, Cat. No. #RNSCL‐C; Cosmo Bio, Tokyo, Japan) was cultured as previously described., , , To differentiate 1464R cells into neuronal and glial cells, dissociated 1464R cells were seeded on poly‐ l ‐lysine (PLL; #P1524; Sigma‐Aldrich, St Louis, MO, USA)‐coated 12‐well plates or 9‐mm ACLAR round coverslips (Allied Fibers & Plastics, Pottsville, PA, USA) at a density of 4–6 × 10 5 cells per well and 1–2 × 10 4 cells per coverslip, respectively, and maintained in differentiation medium composed of F12 medium (Cat. No. #11765‐054; Thermo Fisher Scientific, Waltham, CA, USA) containing 5% fetal bovine serum (Moregate, Bulimba, Australia), 0.5% N2 supplement (Cat. No. #17502‐048; Thermo Fisher), 1% B27 supplement (Cat. No. #17504‐044; Thermo Fisher), 1 μ m all‐trans retinoic acid (ATRA; Cat. No. # R2625; Sigma‐Aldrich), 50 units/mL penicillin and 50 μg/mL streptomycin in 5% CO 2 at 37°C.

Techniques: Western Blot, Derivative Assay, Infection, Expressing

Journal: Neuropathology

Article Title: Praja1 RING ‐finger E3 ubiquitin ligase is a common suppressor of neurodegenerative disease‐associated protein aggregation

doi: 10.1111/neup.12840

Figure Lengend Snippet: Suppression of the aggregate formation of neurodegenerative disease‐associated proteins by HSF1. (A) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human wild‐type (WT) and C‐terminal fragment (CTF) TDP‐43, WT and P525L FUS, WT and G93A SOD1, WT and S53T α‐synuclein, ataxin‐3 Q28 and G84, and huntingtin exon 1 Q23 and Q74 (arrowheads), and EGFP‐tagged HSF1 in the presence of 0.5 μ m MG‐132. (B) Densitometric analysis of FLAG Western blot data of RIPA‐insoluble fractions versus PJA1 (arrowheads in A; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples. Results are presented as mean ± SD. All bars; P < 0.05. (C) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing EGFP‐tagged HSF1 and rat negative control (NC) or PJA1 shRNA/EGFP (AxshNC/EGFP, AxshPJA1/EGFP) in the presence of 0.5 μ m MG‐132. (D) Densitometric analysis of PJA1 Western blot data (arrowheads in C; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (* P < 0.05). (E) Western blot analysis of 1464R‐derived neuronal cells infected with adenoviruses expressing DsRed‐ and FLAG‐tagged human WT and CTF TDP‐43, EGFP‐tagged HSF1 and rat NC or PJA1 shRNA/EGFP in the presence of 0.5 μ m MG‐132. (F) Densitometric analysis of FLAG Western blot data of RIPA‐insoluble WT and CTF TDP‐43 (arrowheads in E; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP‐treated control samples in the presence of MG132. Results are presented as mean ± SD (N/S; not significant).

Article Snippet: The adult rat neural stem cell line 1464R that we have established (Cat. No. #T0746; Applied Biological Materials, Canada, Cat. No. #RNSCL‐C; Cosmo Bio, Tokyo, Japan) was cultured as previously described., , , To differentiate 1464R cells into neuronal and glial cells, dissociated 1464R cells were seeded on poly‐ l ‐lysine (PLL; #P1524; Sigma‐Aldrich, St Louis, MO, USA)‐coated 12‐well plates or 9‐mm ACLAR round coverslips (Allied Fibers & Plastics, Pottsville, PA, USA) at a density of 4–6 × 10 5 cells per well and 1–2 × 10 4 cells per coverslip, respectively, and maintained in differentiation medium composed of F12 medium (Cat. No. #11765‐054; Thermo Fisher Scientific, Waltham, CA, USA) containing 5% fetal bovine serum (Moregate, Bulimba, Australia), 0.5% N2 supplement (Cat. No. #17502‐048; Thermo Fisher), 1% B27 supplement (Cat. No. #17504‐044; Thermo Fisher), 1 μ m all‐trans retinoic acid (ATRA; Cat. No. # R2625; Sigma‐Aldrich), 50 units/mL penicillin and 50 μg/mL streptomycin in 5% CO 2 at 37°C.

Techniques: Western Blot, Derivative Assay, Infection, Expressing, Control, Negative Control, shRNA