Journal: PLoS ONE

Article Title: Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

doi: 10.1371/journal.pone.0048246

Figure Lengend Snippet: Cell surface (a) and extracellular (b) ANXA1 from C2C12 cells in GM (0 differentiation day) and after exposure for the indicated times (3, 5, and 7 differentiation days) to DM was analyzed by Western blot with anti-ANXA1 (a, b) antibody. Total cell protein extracts were analyzed by Western blot with anti-ANXA1 (c) and with anti-MyoD (d), anti-Myogenin (e), and anti-MyHC (f) antibodies to assess myogenic differentiation rate. The protein bands were normalized on tubulin levels (g). The data are representative of 5 experiments with similar results.

Article Snippet: Treatments with ionomycin (1 mM) and/or fMLP (50 nM; Sigma-Aldrich), ANXA1 N-terminal peptide Ac2-26 (100 nM; Tocris Biosciences), cyclosporine H (CsH; 500 nM; Alexis-Biochemicals) were carried out by adding the appropriate concentrations of each substance into the cuvette in Ca 2+ -free HBSS/0.5 mM EDTA buffer.

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

doi: 10.1371/journal.pone.0048246

Figure Lengend Snippet: ( A ) Results for control, fMLP, Ac2-26, CsH, fMLP + CsH and Ac2-26+ CsH are reported as means of three experiments, measuring individual cell migrations at different times. Bars represent standard errors. ( B ) Cytosolic, cell surface and extracellular ANXA1 from C2C12 cells after exposure for the indicated time (16 h) to fMLP, Ac2-26, CsH, Ac2-26+ CsH and fMLP + CsH were analyzed by Western blot with anti-ANXA1 and anti-tubulin antibodies. The data are representative of 5 experiments with similar results. *** <0.001 vs control; §§§ <0.001 vs Ac 2-26 or fMLP.

Article Snippet: Treatments with ionomycin (1 mM) and/or fMLP (50 nM; Sigma-Aldrich), ANXA1 N-terminal peptide Ac2-26 (100 nM; Tocris Biosciences), cyclosporine H (CsH; 500 nM; Alexis-Biochemicals) were carried out by adding the appropriate concentrations of each substance into the cuvette in Ca 2+ -free HBSS/0.5 mM EDTA buffer.

Techniques: Control, Western Blot

Journal: PLoS ONE

Article Title: Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

doi: 10.1371/journal.pone.0048246

Figure Lengend Snippet: Proteins identified as possible Ac2-26 partners by chemical proteomics.

Article Snippet: Treatments with ionomycin (1 mM) and/or fMLP (50 nM; Sigma-Aldrich), ANXA1 N-terminal peptide Ac2-26 (100 nM; Tocris Biosciences), cyclosporine H (CsH; 500 nM; Alexis-Biochemicals) were carried out by adding the appropriate concentrations of each substance into the cuvette in Ca 2+ -free HBSS/0.5 mM EDTA buffer.

Techniques:

Journal: PLoS ONE

Article Title: Annexin A1 Induces Skeletal Muscle Cell Migration Acting through Formyl Peptide Receptors

doi: 10.1371/journal.pone.0048246

Figure Lengend Snippet: Cultured murine C2C12 myoblasts fixed and labeled with fluorescent antibody against ANXA1 and with FITC-conjugated Phalloidin in Ca 2+ -free conditions (a, c, e, g) and in a medium containing 2 mM Ca 2+ (b, d, f, h). (B) An increase of intracellular Ca 2+ levels leads to ANXA1 relocation to the plasma membrane (a); at high Ca 2+ concentrations actin filaments (green) are significantly damaged by high intracellular increase of Ca 2+ level (e): in this condition ANXA1(red) is strikingly shifted to the plasma membrane (d). Bar = 25 µm.

Article Snippet: Treatments with ionomycin (1 mM) and/or fMLP (50 nM; Sigma-Aldrich), ANXA1 N-terminal peptide Ac2-26 (100 nM; Tocris Biosciences), cyclosporine H (CsH; 500 nM; Alexis-Biochemicals) were carried out by adding the appropriate concentrations of each substance into the cuvette in Ca 2+ -free HBSS/0.5 mM EDTA buffer.

Techniques: Cell Culture, Labeling, Clinical Proteomics, Membrane

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: HSPB1 is induced by Ac2-26 in microglia. RT-PCR analysis of total RNA isolated from rat brain tissues, neurons, astrocytes and microglial cells revealed the presence of sHSP transcripts (A,B) . BV-2 cells were treated with Ac2-26 (10 μM) before they were continuously exposed to oxygen glucose deprivation (OGD) for 1 h and reperfusion for 24 h. The mRNA expression of HSPB1 was assessed by RT-PCR (C) , and the protein levels were detected by Western blotting (D) as indicated. (E,F) Microglia were treated with NCM or Ac2-26 (10 μM) for 24 h. Protein levels were assessed by Western blotting as indicated. The data are representative of at least three independent experiments and shown as the mean ± SEM. All data were analyzed using one-way ANOVA with Tukey’s multiple comparison tests; Ac2-26 induced HSPB1 protein expression (E) was analyzed using unpaired Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. control. (G) Protein cell lysates from HeLa cells transfected with GFP-ANXA1 were immunoprecipitated with anti-GFP mAb or mouse IgG and immunoblotted using an anti-HSPB1 mAb. This experiment was repeated at least three times.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Western Blot, Comparison, Control, Transfection, Immunoprecipitation

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: Interaction between HSPB1 and IKKβ. (A) The interaction between HSPB1 and IKKβ in microglia was assayed by CO-IP. Microglia were incubated with NCM in the presence or absence of Ac2-26 (10 μM) for 24 h. Cell lysates were immunoprecipitated with anti-HSPB1 and immunoblotted with anti-IKKβ. (B) HSPB1 overexpression reduced IKKβ levels. HeLa or BV-2 cells were transfected with vectors expressing GFP-tagged HSPB1 (7.5 μg/well) or FLAG-tagged IKKβ (7.5 μg/well) as indicated. Whole cell lysates were detected with anti-FLAG and anti-GFP. (C) Fold change of the IKKβ/β-actin ratio in the indicated conditions. (D) The expression of HSPB1 was knocked down in microglia using adenovirus carrying HSPB1-specific shRNA as described in the “Materials and Methods” section but not in cells transfected with control shNC. (E,F) Microglia were transfected with adenovirus carrying shRNA against HSPB1. At 48 h post-transfection, IKKβ protein levels were detected by immunoblotting (E) , and TNF-α production was assessed by ELISA (F) . Microglial cells transfected with shHSPB1 were then treated with Ac2-26 (10 μM), and protein levels were measured by Western blotting (G) as indicated. The data are representative of 3–7 independent experiments. Throughout the figure, the error bars represent the mean ± SEM. Data were analyzed using one-way ANOVA with Tukey’s multiple comparison tests between three groups and unpaired Student’s t test with two groups. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 vs. control.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation, Over Expression, Transfection, Expressing, shRNA, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: ANXA1 and HSPB1 transport IKKβ into lysosomes. (A) Amino acid locations of the putative CMA-targeting motifs in IKKβ, ANXA1 (Ac2-26) and HSPB1 proteins. (B) HeLa cells were immunoprecipitated with anti-Hsc70 and immunoblotted with anti- IKKβ, ANXA1, HSPB1 and Hsc70 antibodies. (C) LAMP-2A interacts with IKKβ, ANXA1 and HSPB1. HeLa cells were transfected with the indicated plasmids (100 μg each per 100-mm dish). The interaction was assessed by IP with anti-HA, followed by IP of Flag or GFP as indicated. (D) Silencing Hsc70 disrupted the binding of LAMP-2A and IKKβ. Anti-LAMP-2A immunoprecipitates from HeLa cells transfected with control or Hsc70 shRNA were immunoblotted with anti-IKKβ, ANXA1 and HSPB1 antibodies. (E) Anti-LAMP-2A immunoprecipitates from HeLa cells were transfected with control ANXA1 shRNA or adenovirus carrying HSPB1-specific shRNA and immunoblotted with anti-IKKβ, ANXA1 and HSPB1 antibodies. All data are representative of 3–4 independent experiments.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Immunoprecipitation, Transfection, Binding Assay, Control, shRNA

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: IKKβ is degraded by CMA. (A) Linearity of dye uptake with increasing amounts of lysosomal protein. (B) Homogenates (Homo) and lysosomes isolated from rat brain tissues were pretreated with chloroquine (50 μM) for 30 min at 4°C prior to the addition of Ac2-26(1 mg/g) for an additional 30 min. A total of 100 μg of protein was then processed and analyzed by Western blotting as indicated. (C) The protein levels of IKKβ, ANXA1, and HSPB1 per 100 μg of total protein from rat brain tissues. (D) Microglia were transfected with adenovirus carrying shRNA against LAMP-2A. LAMP-2A levels were examined by Western blotting. (E,F) Microglial cells were transfected with LAMP-2A shRNA. The supernatants were assessed for TNF-α by ELISA (E) , and the cell lysates were analyzed with antibodies to IKKβ and β-actin by Western blotting (F) . Microglial cells transfected with shLAMP-2A were then treated with Ac2-26, and protein levels were measured by Western blotting (G) as indicated. The data are representative of 3–6 independent experiments and were analyzed by one-way ANOVA with Tukey’s multiple comparison tests between three groups and unpaired Student’s t test between two groups. Throughout the figure, the error bars represent the mean ± SEM. * P < 0.05; ** P < 0.01 vs. control. (H) Microglia transfected with shHSPB1 or shLAMP-2A for 48 h in the presence or absence of NCM for 24 h. The expression levels of IKKβ mRNA were determined by real-time quantitative PCR and normalized to β-actin. The levels of untreated control cells (CON) were set to100%. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison tests.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Isolation, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Comparison, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: Proposed mechanism of Ac2-26-induced CMA-dependent degradation of IKKβ. Ac2-26 enters microglial cells to bind to and induce HSPB1, and HSPB1 further associates with IKKβ. The Ac2-26-HSPB1-IKKβ protein complex is recruited by a chaperone complex containing Hsc70 and binds to LAMP-2A on lysosomes. Ac2-26 and HSPB1 then assist with the translocation of IKKβ through LAMP-2A multimers into lysosomes for degradation.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Translocation Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: HSPB1 is induced by Ac2-26 in microglia. RT-PCR analysis of total RNA isolated from rat brain tissues, neurons, astrocytes and microglial cells revealed the presence of sHSP transcripts (A,B) . BV-2 cells were treated with Ac2-26 (10 μM) before they were continuously exposed to oxygen glucose deprivation (OGD) for 1 h and reperfusion for 24 h. The mRNA expression of HSPB1 was assessed by RT-PCR (C) , and the protein levels were detected by Western blotting (D) as indicated. (E,F) Microglia were treated with NCM or Ac2-26 (10 μM) for 24 h. Protein levels were assessed by Western blotting as indicated. The data are representative of at least three independent experiments and shown as the mean ± SEM. All data were analyzed using one-way ANOVA with Tukey’s multiple comparison tests; Ac2-26 induced HSPB1 protein expression (E) was analyzed using unpaired Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. control. (G) Protein cell lysates from HeLa cells transfected with GFP-ANXA1 were immunoprecipitated with anti-GFP mAb or mouse IgG and immunoblotted using an anti-HSPB1 mAb. This experiment was repeated at least three times.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Expressing, Western Blot, Comparison, Control, Transfection, Immunoprecipitation

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: Interaction between HSPB1 and IKKβ. (A) The interaction between HSPB1 and IKKβ in microglia was assayed by CO-IP. Microglia were incubated with NCM in the presence or absence of Ac2-26 (10 μM) for 24 h. Cell lysates were immunoprecipitated with anti-HSPB1 and immunoblotted with anti-IKKβ. (B) HSPB1 overexpression reduced IKKβ levels. HeLa or BV-2 cells were transfected with vectors expressing GFP-tagged HSPB1 (7.5 μg/well) or FLAG-tagged IKKβ (7.5 μg/well) as indicated. Whole cell lysates were detected with anti-FLAG and anti-GFP. (C) Fold change of the IKKβ/β-actin ratio in the indicated conditions. (D) The expression of HSPB1 was knocked down in microglia using adenovirus carrying HSPB1-specific shRNA as described in the “Materials and Methods” section but not in cells transfected with control shNC. (E,F) Microglia were transfected with adenovirus carrying shRNA against HSPB1. At 48 h post-transfection, IKKβ protein levels were detected by immunoblotting (E) , and TNF-α production was assessed by ELISA (F) . Microglial cells transfected with shHSPB1 were then treated with Ac2-26 (10 μM), and protein levels were measured by Western blotting (G) as indicated. The data are representative of 3–7 independent experiments. Throughout the figure, the error bars represent the mean ± SEM. Data were analyzed using one-way ANOVA with Tukey’s multiple comparison tests between three groups and unpaired Student’s t test with two groups. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 vs. control.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Co-Immunoprecipitation Assay, Incubation, Immunoprecipitation, Over Expression, Transfection, Expressing, shRNA, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: ANXA1 and HSPB1 transport IKKβ into lysosomes. (A) Amino acid locations of the putative CMA-targeting motifs in IKKβ, ANXA1 (Ac2-26) and HSPB1 proteins. (B) HeLa cells were immunoprecipitated with anti-Hsc70 and immunoblotted with anti- IKKβ, ANXA1, HSPB1 and Hsc70 antibodies. (C) LAMP-2A interacts with IKKβ, ANXA1 and HSPB1. HeLa cells were transfected with the indicated plasmids (100 μg each per 100-mm dish). The interaction was assessed by IP with anti-HA, followed by IP of Flag or GFP as indicated. (D) Silencing Hsc70 disrupted the binding of LAMP-2A and IKKβ. Anti-LAMP-2A immunoprecipitates from HeLa cells transfected with control or Hsc70 shRNA were immunoblotted with anti-IKKβ, ANXA1 and HSPB1 antibodies. (E) Anti-LAMP-2A immunoprecipitates from HeLa cells were transfected with control ANXA1 shRNA or adenovirus carrying HSPB1-specific shRNA and immunoblotted with anti-IKKβ, ANXA1 and HSPB1 antibodies. All data are representative of 3–4 independent experiments.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Immunoprecipitation, Transfection, Binding Assay, Control, shRNA

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: IKKβ is degraded by CMA. (A) Linearity of dye uptake with increasing amounts of lysosomal protein. (B) Homogenates (Homo) and lysosomes isolated from rat brain tissues were pretreated with chloroquine (50 μM) for 30 min at 4°C prior to the addition of Ac2-26(1 mg/g) for an additional 30 min. A total of 100 μg of protein was then processed and analyzed by Western blotting as indicated. (C) The protein levels of IKKβ, ANXA1, and HSPB1 per 100 μg of total protein from rat brain tissues. (D) Microglia were transfected with adenovirus carrying shRNA against LAMP-2A. LAMP-2A levels were examined by Western blotting. (E,F) Microglial cells were transfected with LAMP-2A shRNA. The supernatants were assessed for TNF-α by ELISA (E) , and the cell lysates were analyzed with antibodies to IKKβ and β-actin by Western blotting (F) . Microglial cells transfected with shLAMP-2A were then treated with Ac2-26, and protein levels were measured by Western blotting (G) as indicated. The data are representative of 3–6 independent experiments and were analyzed by one-way ANOVA with Tukey’s multiple comparison tests between three groups and unpaired Student’s t test between two groups. Throughout the figure, the error bars represent the mean ± SEM. * P < 0.05; ** P < 0.01 vs. control. (H) Microglia transfected with shHSPB1 or shLAMP-2A for 48 h in the presence or absence of NCM for 24 h. The expression levels of IKKβ mRNA were determined by real-time quantitative PCR and normalized to β-actin. The levels of untreated control cells (CON) were set to100%. Data were analyzed by one-way ANOVA with Tukey’s multiple comparison tests.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Isolation, Western Blot, Transfection, shRNA, Enzyme-linked Immunosorbent Assay, Comparison, Control, Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Molecular Neuroscience

Article Title: Ac2-26 Induces IKKβ Degradation Through Chaperone-Mediated Autophagy Via HSPB1 in NCM-Treated Microglia

doi: 10.3389/fnmol.2018.00076

Figure Lengend Snippet: Proposed mechanism of Ac2-26-induced CMA-dependent degradation of IKKβ. Ac2-26 enters microglial cells to bind to and induce HSPB1, and HSPB1 further associates with IKKβ. The Ac2-26-HSPB1-IKKβ protein complex is recruited by a chaperone complex containing Hsc70 and binds to LAMP-2A on lysosomes. Ac2-26 and HSPB1 then assist with the translocation of IKKβ through LAMP-2A multimers into lysosomes for degradation.

Article Snippet: LAMP-2A and HSPB1 expression was assessed by immunoblot analysis with antibodies as indicated. pEGFP-hsp27 wt FL was a gift from Andrea Doseff (Addgene plasmid #17444); pcDNA-IKKβ-FLAG WT was a gift from Warner Greene (Addgene plasmid #23298); pEGFP-ANXA1 wt was kindly supplied by Li et al. ( ); pEGFP-ANXA1-N and Flag-Ac2-26 were constructed with standard procedures; HA-LAMP-2A WT was purchased from Vigene Biosciences (Shandong, China).

Techniques: Translocation Assay