Journal:

Article Title: Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs

doi: 10.1128/AAC.46.12.3817-3822.2002

Figure Lengend Snippet: Antimicrobial susceptibility profiles of E. aerogenes strains 810, 810-REV, and ATCC 13048

Article Snippet: Since the MICs of imipenem, meropenem, and cefepime were much lower for the imipenem-susceptible revertant than for parent strain 810 and resistance did not appear to be β-lactamase mediated, we examined strains 810 and 810-REV for alterations in OMP expression and compared their OMP expression to the OMP expression of E. aerogenes type strain ATCC 13048 (Fig. and ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 3. caption a7 Resolution of OMPs from E. aerogenes type strain ATCC 13048, E. aerogenes 810, and E. aerogenes 810-REV on 4 to 16% (A) and 4 to 12% (B) Tris-glycine-SDS gels and 4 to 12% NuPAGE gels (C).

Techniques:

Journal:

Article Title: Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs

doi: 10.1128/AAC.46.12.3817-3822.2002

Figure Lengend Snippet: Resolution of OMPs from E. aerogenes type strain ATCC 13048, E. aerogenes 810, and E. aerogenes 810-REV on 4 to 16% (A) and 4 to 12% (B) Tris-glycine-SDS gels and 4 to 12% NuPAGE gels (C). Strain 810 was grown in the presence and absence of imipenem. The closed arrowheads indicate the 42-kDa porin (presumed OmpC analog), and the open arrowheads indicate the 39-kDa porin (presumed OmpF analog).

Article Snippet: Since the MICs of imipenem, meropenem, and cefepime were much lower for the imipenem-susceptible revertant than for parent strain 810 and resistance did not appear to be β-lactamase mediated, we examined strains 810 and 810-REV for alterations in OMP expression and compared their OMP expression to the OMP expression of E. aerogenes type strain ATCC 13048 (Fig. and ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 3. caption a7 Resolution of OMPs from E. aerogenes type strain ATCC 13048, E. aerogenes 810, and E. aerogenes 810-REV on 4 to 16% (A) and 4 to 12% (B) Tris-glycine-SDS gels and 4 to 12% NuPAGE gels (C).

Techniques:

Journal:

Article Title: Carbapenem Resistance in a Clinical Isolate of Enterobacter aerogenes Is Associated with Decreased Expression of OmpF and OmpC Porin Analogs

doi: 10.1128/AAC.46.12.3817-3822.2002

Figure Lengend Snippet: SDS-polyacrylamide gel electrophoresis and Western blot analysis of OMPs from E. aerogenes 810, carbapenem-susceptible revertant 810-REV, and E. aerogenes ATCC 13048 (control strain). (A) Analysis of OMPs on a 4 to 12% Tris-glycine-SDS gel; (B) Western blot analysis of OMPs with polyclonal anti-OmpK35 antiserum (the OmpK35 antibody cross-reacts with OmpK36 [16]); (C) Western blot analysis of OMPs with polyclonal anti-OmpK36 antiserum. The arrows with the white heads indicate the OmpC (OmpK36) analogs; the arrows with black heads indicate the OmpF (OmpK35) analogs.

Article Snippet: Since the MICs of imipenem, meropenem, and cefepime were much lower for the imipenem-susceptible revertant than for parent strain 810 and resistance did not appear to be β-lactamase mediated, we examined strains 810 and 810-REV for alterations in OMP expression and compared their OMP expression to the OMP expression of E. aerogenes type strain ATCC 13048 (Fig. and ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window FIG. 3. caption a7 Resolution of OMPs from E. aerogenes type strain ATCC 13048, E. aerogenes 810, and E. aerogenes 810-REV on 4 to 16% (A) and 4 to 12% (B) Tris-glycine-SDS gels and 4 to 12% NuPAGE gels (C).

Techniques: Polyacrylamide Gel Electrophoresis, Western Blot, SDS-Gel

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: Identification of UBE2O as a SMAD6-interacting partner. (A) Proteomic screening of interacting protein for SMAD6. HEK293T cells transfected with TAP-SMAD6 were lysed for immunoprecipitation. Purified protein complexes were separated by SDS–PAGE and stained by colloidal Coomassie Blue. Triangle indicates accumulation of SMAD6. Blue or grey ovals in right panel indicate the known or unknown interacting protein for SMAD6, UBE2O is highlighted in yellow. The graph was created using Cytoscape's Edge-Weighted Spring Embedded layout using the Fisher exact P-values as weights, such that more significant interactors will tend to be closer to SMAD6. Thicker edges represent higher significance (Smoot et al, 2011). (B, C) Interaction between UBE2O and SMAD6 or SMAD7. HEK293T cells were transfected with Flag-SMAD6, Flag-SMAD7 alone or with UBE2O-Myc as indicated for 48 h. Cells were harvested for immunoprecipitation with anti-Flag (B) or anti-Myc (C) resin. Myc (B) or Flag (C) antibody was used to detect the interaction. (D, E) UBE2O is a SMAD6-interacting protein. HEK293T cells were transfected with C-terminal- or N-terminal-Flag-tagged UBE2O (D) or Flag-tagged SMAD6 (E) for 48 h. Cells were harvested for immunoprecipitation with anti-Flag resin. SMAD6 (D) or UBE2O (E) antibody was used to detect the interaction. (F) The N-terminal of UBE2O interacts with SMAD6. (Top panel) Schematic illustration of the deletion mutants of UBE2O. (Bottom panel) HEK293T cells were transfected with Flag-SMAD6, UBE2O-Myc, or its deletions as indicated for 48 h. Cells were harvested for immunoprecipitation with anti-Flag resin. Myc antibody was used to detect the interaction. (G) The MH2 domain of SMAD6 interacts with UBE2O. (Top panel) Schematic illustration of the deletion mutants of SMAD6. (Bottom panel) HEK293T cells were transfected with Flag-SMAD6, SMAD6 deletions with UBE2O-Myc as indicated for 48 h. Cells were harvested for immunoprecipitation with anti-Flag resin. Myc antibody was used to detect the interaction. IP, immunoprecipitation; IB, immunoblot; TCL, total cell lysate.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Transfection, Immunoprecipitation, Purification, SDS Page, Staining, Western Blot

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: UBE2O monoubiquitinates SMAD6. (A) UBE2O specifically ubiquitinates SMAD6 and SMAD7. HEK293T cells were transfected with indicated Flag-SMADs and HA-ubiquitin with or without UBE2O-Myc for 48 h. In vivo ubiquitination of SMADs proteins was performed by immunoprecipitation. Cells were sonicated with 1% SDS, then diluted to 0.1% SDS for immunoprecipitation with anti-Flag resin. HA antibody was used to detect ubiquitinated SMADs. (B, C) UBE2O monoubiquitinates SMAD6 in vivo. HEK293T cells were transfected with Flag-SMAD6, HA-ubiquitin (B) or His-ubiquitin (C) and UBE2O-Myc for 48 h. (B) In vivo ubiquitination of SMAD6 by immunoprecipitation was performed as described in panel A. Monoubiquitinated SMAD6 was detected by HA or FK2 (specifically recognise ubiquitinated protein) antibody. (C) Cells were lysed in 8 M urea buffer, and nickel affinity resin was used to perform the pull down (Ni-NTA); monoubiquitinated SMAD6 was detected by Flag antibody. Asterisk indicates input of SMAD6. (D) Depletion of UBE2O decreases monoubiquitination of SMAD6 in vivo. HEK293T cells stably expressing UBE2O shRNA (shUBE2O-1, -2) were transfected with Flag-SMAD6 and His-ubiquitin for 48 h. Nickel pull down was performed as described in Figure 2C. Monoubiquitinated SMAD6 (ubi-SMAD6) was detected by Flag antibody. Asterisk indicates input of SMAD6.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Transfection, Ubiquitin Proteomics, In Vivo, Immunoprecipitation, Sonication, Stable Transfection, Expressing, shRNA

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: UBE2O functions as an E2-E3 hybrid to monoubiquitinate SMAD6. (A) Cysteine 885 is the E2 active site of UBE2O for monoubiquitinating SMAD6. The conserved and putative E2 active site cysteine 885, located in the UBC domain of UBE2O, was mutated to serine. HEK293T cells were transfected with Flag-SMAD6, His-ubiquitin, and UBE2O-Myc or C885S-Myc for 48 h. Cells were lysed in 8 M urea buffer, and nickel affinity resin was used to perform the pull down. Flag antibody was used to detect monoubiquitinated SMAD6. Asterisk indicates input SMAD6. (B) UBE2O functions as an E2-E3 hybrid in SMAD6 monoubiquitination. Purified Flag-SMAD6, UBE2O-Myc or UBE2O deletions (described in Figure 1F) with Myc tag were incubated with E1 enzyme and HA-ubiquitin as described in Materials and Methods. HA antibody was used to detect monoubiquitinated SMAD6 after immunoprecipitation with anti-Flag resin.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Transfection, Ubiquitin Proteomics, Purification, Incubation, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: Lysine 174 of SMAD6 is monoubiquitinated by UBE2O. (A) Schematic of the chimeric SMAD6. Wild-type (wt) and lysine-deficient (k0) SMAD6 were used to make chimeric SMAD6. SMAD6 was divided into four regions and chimeric SMAD6 was named according to presence of lysine. Example: W1L234 was made by fusing region 1 of wt SMAD6 and domain 2, 3 and 4 of k0 SMAD6. (B) Lysine is responsible for the monoubiquitination of SMAD6 by UBE2O. HEK293T cells were transfected with wild-type Flag-SMAD6 (wt) or lysine-deficient SMAD6 (k0), His-ubiquitin, and UBE2O-Myc for 48 h. Cells were lysed in 8 M urea buffer, and nickel affinity resin was used to perform the pull down. Flag antibody was used to detect monoubiquitinated SMAD6. Triangle shows the unspecific band when lysine-deficient SMAD6 was co-transfected with ubiquitin, which could be the N-terminal ubiquitination when all lysine were mutated in SMAD6. (C) N-terminal of SMAD6 is monoubiquitinated by UBE2O. HEK293T cells were transfected with the indicated Flag-SMAD6 (wild-type or chimeric SMAD6), Myc-His-ubiquitin, and UBE2O. Nickel pull down (described in Figure 4B) or immunoprecipitation with anti-Flag (described in Figure 2A) resin was used to identify the monoubiquitination region of SMAD6. (D) UBE2O monoubiquitinates lysine 174 of SMAD6. HEK293T cells were transfected with wild-type Flag-SMAD6 (wt), lysine-deficient SMAD6 (k0), or the indicated single- or double-mutated SMAD6, His-ubiquitin, and UBE2O for 48 h. Nickel pull down was performed as in Figure 4B.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Transfection, Ubiquitin Proteomics, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: Monoubiquitination of SMAD6 potentiates BMP7-induced signalling. (A) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced signalling. Analysis of BMP7-induced signalling activation in HEPG2 cells with co-transfection of SMAD6 (wt), lysine 182 or 174 to arginine (K182R or K174R, left panel) or increase amounts of lysine 174 to arginine (K174R, right panel). Data from triplicates are presented as the mean ±s.d. of a representative experiment. (B) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced SMAD1 phosphorylation. C3H10T1/2 cells stably expressing empty vector, SMAD6, or K174R were stimulated with BMP7 at the indicated time points, and western blots were used to analyse phosphorylated SMAD1 level (pSMAD1). (C) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced adipocyte differentiation. C3H10T1/2 cells stably expressing empty vector, SMAD6, or K174R were pre-treated with BMP7 for 3 days and differentiated into adipocytes for another 8 days. Cells were fixed and stained with Oil Red O as described in Materials and Methods. Whole well and higher magnification are shown. (D) Lysine 174-mutated SMAD6 increases the inhibitory ability of SMAD6 on BMP7-induced adipocyte-related genes expression. C3H10T1/2 cells stably expressing empty vector, SMAD6, or K174R were pre-treated with BMP7 for 3 days and differentiated into adipocytes for another 8 days. Adi(day) represents adipocyte differentiation days. Adipocyte cell markers AP2, CEBP/α, PGC1β, and PPARγ were analysed by real-time PCR. *indicates P<0.05 and **indicates P<0.01 (wild-type SMAD6 was compared with empty vector and K174R mutated SMAD6 was compared with wild-type SMAD6). (E) SMAD6 and UBE2O are upregulated in BMP7-induced commitment of mesenchymal stem cells to proliferating pre-adipocytes. C3H10T1/2 cells were pre-treated with 100 ng/μl BMP7 in DMEM supplemented with 10% FBS for 3 days to reach confluence, cells were induced to adipocytes as described in Materials and Methods. Cells were harvested for western blot at the indicated time points. Adi(day) represents adipocyte differentiation days. (F) The relative protein level of UBE2O or SMAD6 (compared with protein level of UBE2O or SMAD6 without BMP7 treatment) was quantified. *indicates P<0.05.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Activation Assay, Cotransfection, Phospho-proteomics, Stable Transfection, Expressing, Plasmid Preparation, Western Blot, Staining, Real-time Polymerase Chain Reaction

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: Monoubiquitinated SMAD6 restrains the interaction of ALK2 and SMAD6. (A) Subcellular localisation of monoubiquitinated SMAD6. HEK293T cells were transfected with Flag-SMAD6, HA-ubiquitin, and UBE2O-Myc for 48 h. Cell fractionation assays were performed as described in Materials and Methods. Four endogenous proteins (p65 for cytoplasmic extract, N-cadherin for membrane extract, HDAC2 for soluble nuclear extract and phosphorylated Histone 3 for chromatin-bound extract) were used as markers for cell fractionation assay. In vivo ubiquitination of fractionated SMAD6 was performed by immunoprecipitation with Flag-resin. HA antibody was used to detect monoubiquitinated SMAD6. CE, cytoplasmic extract; ME, membrane extract; NE, soluble nuclear extract; CB, chromatin-bound extract. (B, C) Monoubiquitinated SMAD6 reduces the interaction between SMAD6 and caALK2. Purified Flag-SMAD6, Flag-ubiquitinated SMAD6 (F-Ubi-SMAD6), and HA-caALK2 from HEK293T cells were mixed together as indicated, and results of immunoprecipitation with HA- (B) or Flag- (C) resin were analysed with indicated antibodies. Asterisks indicate the input.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Transfection, Ubiquitin Proteomics, Cell Fractionation, Membrane, In Vivo, Immunoprecipitation, Purification

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: UBE2O potentiates BMP7-induced SMAD signalling. (A, C) UBE2O positively regulates BMP7-induced signalling activation. Analysis of BMP7-induced signalling activation in HEPG2 cells with knockdown of UBE2O (A), or with increased amounts of UBE2O or C885S-mutated UBE2O (C). Data from triplicates are presented as the mean ±s.d. of a representative experiment. NS represents non-specific shRNA. (B, D) UBE2O potentiates BMP7-induced SMAD1 phosphorylation. C3H10T1/2 cells stably expressing UBE2O shRNA (shUBE2O-1, -2) (B) or stably expressing UBE2O or C885S-mutated UBE2O (D) were stimulated with BMP7 at the indicated time points. Western blots were used to analyse phosphorylated SMAD1 level with indicated antibodies. (E, F) UBE2O potentiates BMP7-induced adipocyte differentiation. C3H10T1/2 cells stably expressing empty vector, UBE2O, or C885- mutated UBE2O were pre-treated with BMP7 for 3 days and differentiated into adipocytes for another 8 days. Cells were fixed and stained with Oil Red O as described in the Materials and Methods (E) or analysed by real-time PCR for adipocyte markers AP2, CEBP/α, PGC1β, and PPARγ (F). Adi(day) represents adipocyte differentiation days. *indicates P<0.05 and **indicates P<0.01(wild-type UBE2O was compared with empty vector and C885S-mutated UBE2O was compared with wild-type UBE2O). (G) UBE2O potentiates BMP6-induced SMAD1 phosphorylation. C2C12 cells stably expressing empty vector or UBE2O-Myc were stimulated with 50 ng/ml BMP6 at the indicated time points. Western blots were used to analyse phosphorylated SMAD1 level with indicated antibodies. (H) UBE2O potentiates BMP6-induced osteoblast-like cell differentiation. C2C12 cells stably expressing empty vector or UBE2O-Myc were treated with 50 ng/ml BMP6 for 3 days, and then cells were harvested for histochemical staining to determine ALP activity. Whole well and higher magnification are shown. (I) The potentiated BMP7-induced signalling by UBE2O depends on SMAD6. Immortalised SMAD6-knockout MEF cells were infected with empty vector or UBE2O-Myc expression viruses. Cells were treated with BMP7 at the indicated time points. Phosphorylated SMAD1 level was analysed by western blot. (J) The relative BMP7-induced SMAD1 phosphorylation level in Figure 7G (normalised to SMAD1) was quantified. **indicates P<0.01, n.s. indicates no significant difference.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Activation Assay, Knockdown, shRNA, Phospho-proteomics, Stable Transfection, Expressing, Western Blot, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Cell Differentiation, Activity Assay, Knock-Out, Infection

Journal: The EMBO Journal

Article Title: Fine-tuning BMP7 signalling in adipogenesis by UBE2O/E2-230K-mediated monoubiquitination of SMAD6

doi: 10.1038/emboj.2013.38

Figure Lengend Snippet: Schematic shows how UBE2O potentiates BMP7 signalling. Without monoubiquitination, SMAD6 binds to activated type I receptor to inhibit BMP7-induced SMAD1 phosphorylation and subsequent regulation of transcriptional responses. Monoubiquitinated SMAD6 by UBE2O impairs the association of activated type I receptor with non-modified SMAD6, resulting in more prominent BMP7/SMAD signalling. pi represents phosphorylation and ubi represents ubiquitination.

Article Snippet: Antibodies used in this study were c-Myc (a-14, sc-789, Santa Cruz Biotechnology), HA (Y-11, sc-805, Santa Cruz Biotechnology), Flag (F3165, Sigma), β-actin (A5441, Sigma), UBE2O (NBP1-03336, Novus Biologicals), SMAD6 (9519, Cell Signaling and sc-13048, Santa Cruz Biotechnology), SMAD1 (sc-7965, Santa Cruz Biotechnology, 6944, Cell Signaling and 060653, Upstate), pSMAD1 (9511, Cell Signaling), and FK2 (BML-PW8810, Enzo Life Sciences).

Techniques: Phospho-proteomics, Modification, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A) GPC-4 protein was incubated with either APOE2 or APOE4 proteins at room temperature for 1h, and then separated them by a native gel. Western blot analysis with GPC-4 (left panel) and APOE (right panels) antibodies revealed that combinations of APOE4+GPC-4 were shifted while no observable changes occurred with APOE2+GPC-4. A red arrow in the left panel indicates GPC-4 proteins were shifted when combined with APOE4, but not with APOE2. A red arrow in the right panel indicates APOE4 proteins were shifted when combined with GPC-4, whereas APOE2 proteins were not shifted when combined with GPC-4. ME=β-mercaptoethanol. B, C) Proteins isolated from APOE2/2 and APOE4/4 human brains were co-immunoprecipitated with APOE antibodies (n=3) (B). The levels of immunoprecipitated GPC-4 proteins were normalized with corresponding APOE immunoreactive bands (C). It shows that the APOE/GPC-4 complex is significantly higher in APOE4/4 compared to APOE2/2 . D, E) Representative IHC staining of APOE2/3 (control), APOE2/2 (AD), APOE3/3 (AD) and APOE4/4 (AD) tissues with GFAP and GPC-4 antibodies show that APOE4 carrying AD patients expressed significantly higher levels of GPC-4 in astrocytes compared to control and other APOE genotypes (E). n=5-6. F, G) Astrocytes from APOE4/4 AD brains were grouped into two categories based on the number of the branches at 15μm radius (F). Group 1: less than 10 branches. Group 2: more than 10 branches. Astrocytes with more branches expressed significantly elevated levels of GPC-4 (G). H) AD-associated astrocytic genes were selected from Habib et al, Nature Neuroscience , 2020 and generated a heat map with subtypes of astrocytes from Grubman et al, Nature Neuroscience , 2019. Disease-associated genes were enriched in subtypes 2 and 3, and GPC-4 was enriched in a subtype 3. I-K) Western blot analysis from astrocyte culture shows that treatment with TNF-α and IL-1β significantly increased expression of GPC-4, and it also activated the NF-κB pathway. L-N ) Western blot analysis from astrocyte culture treated with NF-κB pathway blocker, IMD-0354, reversed TNF-α and IL-1β induced expression of GPC-4. n=4. one-way ANOVA or unpaired t-test, **P<0.01, ***P<0.001 and ****P<0.0001. IHC scale bars=20 µm.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Incubation, Western Blot, Isolation, Immunoprecipitation, Immunohistochemistry, Generated, Expressing

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A) Violin blots from Grubman et al, Nature Neuroscience , 2019 show that GPC-4 is expressed in astrocytes. B) An IHC image of 12 days old astrocyte culture with MAP2 and GFAP antibodies show the presence of astrocytes, but not neurons. C, D) Western blot analysis shows that GPC-4 shRNA treated astrocytes expressed approximated 30-40% less GPC-4 proteins compared to control. n=4, unpaired t-test, *P<0.05. IHC scale bars=20 µm.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Western Blot, shRNA

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A-D) Western blot analysis of proteins isolated from neuronal culture, which were treated with GPC-4 protein, shows that GPC-4 significantly enhanced pTau levels (C, D) whereas no changes were observed in total tau protein (B). E, F) Representative IHC staining of GPC-4 treated neuronal culture with AT8 and MAP2 antibodies demonstrates that GPC-4 treatment enhanced pTau levels (E). G) Schematic diagram shows that WT astrocyte-conditioned media (ACM) from astrocyte culture was added to PS19 neuronal culture. H-J) Addition of ACM to neuronal culture increased the pTau (AT8) levels whereas total tau proteins were unaltered. K) Schematic diagram shows that astrocytes were treated with GPC-4 shRNA and the resulting GPC-4 deprived ACM was added to neuronal culture. L-N) Addition of GPC-4 deprived ACM failed to induce tau phosphorylation in neurons. O, P) Representative IHC staining of GPC-4 treated neuronal co-culture ( PS19* MAPT KO animals) shows that GPC-4 treatment enhanced tau spreading/uptake. n=4-5, unpaired t-test, IHC scale bars=20 µm. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Western Blot, Isolation, Immunohistochemistry, shRNA, Co-Culture Assay

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A) IHC staining with GPC-4 antibodies show that GPC-4 shRNA treatment reduced approximately 40% of GPC-4 expression. B) Representative IHC images with anti-GPC-4, GFAP and NeuN antibodies show that injection of CRISPR/dCas9 lentivirus GPC-4 activation system induced the expression of GPC-4 in astrocytes, but not in neurons. C) IHC staining with MC1 antibodies show that CRISPR/dCas9 mediated GPC-4 expression induced tau pathology in vivo . n=3, unpaired t-test, **P<0.01. IHC scale bars=20 µm and 100 µm.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Immunohistochemistry, shRNA, Expressing, Injection, CRISPR, Activation Assay, In Vivo

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A, B) we injected eGFP-2PA-hTauP301L AAV virus in the ipsilateral CA1 regions and control/GPC-4 shRNA in the contralateral CA1 regions, and examined them after 4 weeks. Representative IHC images with GFP and human tau specific antibody Tau13 show that GPC-4 shRNA treatment significantly reduced tau spreading in the contralateral CA1 regions. C, D) In order to induce the expression of GPC-4 proteins, we activated the GPC-4 gene by injecting GPC-4 CRISPR/dCas9 lentivirus activation systems in the cortex or hippocampus. Representative IHC with GPC-4 and GFAP antibodies show that, following a one week of injection, GPC-4 CRISPR/dCas9 robustly induced GPC-4 expression compared to control lentiviral activation particles. E, F) . IHC images with the AT8 antibody show that GPC-4 induced significantly higher levels of pTau in CA1 regions of the hippocampus. G, H) . IHC images with AT8 antibody shows that GPC-4 induced significantly higher levels of pTau in the cortex. n=4-5, unpaired t-test, *P<0.05, **P<0.01 and ***P<0.001. IHC scale bars=20 µm.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Injection, shRNA, Expressing, CRISPR, Activation Assay

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A, B) Cultured neurons (WT) were treated with ACM alone, ACM with APOE4 and GPC-4 deprived (shRNA treated) ACM with APOE4. After 24hrs, neurons were incubated with purified human tau proteins for 1hr, washed several times, and immunostained with human tau antibody (HT-7) to detect human tau proteins. Representative IHC images of APOE4 + ACM treated neuronal culture shows that APOE4 treatment enhanced tau uptake, but in the absence of GPC-4 (GPC-4 shRNA treated ACM) APOE4-induced tau uptake was significantly reduced (B). C, D) we used Tau FRET-biosensor cells to monitor seeding activity of AD-tau proteins in the presence of APOE4 and GPC-4. We incubated Tau FRET-biosensor cells with 0.6μg/ml tau proteins and 1μg/ml APOE4 with/without GPC-4 shRNA for 2 days, and then counted the number of tau inclusion containing cells/area. Compared to tau alone, tau with APOE4 induced significantly higher numbers of tau inclusions in Tau FRET Biosensor cells. Interestingly, APOE4-mediated tau inclusions were reduced in the presence of GPC-4 shRNA. n=8-10. E, F) we injected APOE2 or APOE4 particles isolated from corresponding human brains, in the absence or presence of GPC-4 shRNA. Following 3 weeks of injections, no tau pathology (AT8) was detected with APOE2. APOE4 robustly induced tau pathology, but APOE4 failed to induce tau pathology in the absence of GPC-4 (D). G, H ) IHC staining with MC1 antibodies show that APOE4-mediated tau pathology was diminished in the presence of GPC-4 shRNA. I-M) Human APOE4 iPSCs were differentiated into astrocytes, and the ACM was collected after 3 days of shRNA treatments. The PS19 neurons were treated with ACM for 4 days. Western blot analysis shows that ACM significantly increased AT8 and PHF1 levels, and the ACM-induced tau pathologies were reduced in the absence of GPC-4. n=4-5, one-way ANOVA, **P<0.01, ***P<0.001 and ****P<0.0001. IHC scale bars=20 µm.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Cell Culture, shRNA, Incubation, Purification, Activity Assay, Injection, Isolation, Immunohistochemistry, Western Blot

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A) Western blot analysis with Tau-5 and AT8 antibodies show the isolation of insoluble tau proteins from postmortem AD brains. B, C) GPC-4 shRNA treated Tau FRET-biosensor cells expressed approximated 70% less GPC-4 proteins compared to control. D, E) Western blot analysis shows that GPC-4 shRNA treated iPSCs-astrocytes expressed approximated 80-90% less GPC-4 proteins compared to control. F, G) Western blot analysis shows that GPC-4 shRNA treated iPSCs-astrocytes expressed similar levels of APOE proteins compared to control. n=4-5, unpaired t-test, ***P<0.001.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Western Blot, Isolation, shRNA

Journal: bioRxiv

Article Title: Astrocyte-secreted glypican-4 drives APOE4-dependent tau pathology

doi: 10.1101/2021.07.07.451493

Figure Lengend Snippet: A-C) Western blot analysis from neuronal culture treated with APOE (s) shows that APOE4 significantly enhanced the surface LRP1 (S.LRP1) (C). There were no changes in total LRP1 (T.LRP1) (B). D-F) Western blot analysis from neuronal culture treated with APOE4 or APOE4 with exocytosis inhibitor, Exo-1, show that active exocytosis is required for APOE4-mediated surface trafficking of LRP1. G) Co-immunoprecipitation of human postmortem brain protein samples with LRP1 antibodies and subsequent western blotting revealed that LRP1 and GPC-4 are in the same complex. H-J) Western blot analysis from neuronal culture treated with GPC-4 protein shows that GPC-4 significantly enhanced trafficking of surface LRP1 levels (J), whereas total LRP1 levels were not affected (I). K-M ) Western blot analysis from neuronal culture treated with ACM alone (control), APOE4 + ACM, and with APOE4 + GPC-4 shRNA treated ACM show that APOE4 induced surface expression of LRP1 is mediated through GPC-4. The GPC-4 shRNA treatment reduced the surface expression of LRP1 in the presence of APOE4 (J). N, O) Representative IHC images with AT8 antibodies show that GPC-4 induced tau pathology was reduced in the absence of LRP1. n=4-5, one-way ANOVA or unpaired t-test, **P<0.01, ***P<0.001 and ****P<0.0001. IHC scale bars=20 µm.

Article Snippet: The following primary antibodies were used: AT8 (1:500, #MN1020, Thermo Scientific), AceTau (1:500, #ITK0084, G-Biosciences), MC1 (1:500, a gift from Dr. Peter), MAP2 (1:500, #ab5392, abcam), LRP1 (1:200, #ab92544, abcam), HT-7 (1:200, #MN1000, Thermo Scientific), GPC-4 (1:200, # PA5-72360, 13048-1-AP, Thermo Scientific), GFAP (1:1,000, Aves Labs) and GFP (1:500, # ab290, abcam).

Techniques: Western Blot, Immunoprecipitation, shRNA, Expressing