Journal: Autophagy

Article Title: CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

doi: 10.1080/15548627.2021.1934271

Figure Lengend Snippet: DNM2 or CLTC silencing inhibits HBV replication by blocking endosome formation. (A) Huh7 cells were transfected with si DNM2 or si CLTC and harvested after 48 h. The expression of RAB5A and CD63 was assessed. Scale bar: 10 μm. The fluorescence intensity of RAB5A and CD63 were analyzed using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. (B) Huh7 cells transiently transfected with pSM2 or (C) HepG2.2.15 cells were transfected with si DNM2 or si CLTC , and harvested after 72 h. The expression of DNM2, CLTC, SHBsAg, and HBcAg, the levels of HBsAg and HBeAg in the supernatants, the encapsidated HBV RIs, the levels of intracellular HBV DNA and that in the supernatants, and the HBV RNA levels were measured. (D) Huh7 cells were cotransfected with si DNM2 or si CLTC and pSM2, and harvested after 72 h. The distribution of SHBsAg and HBcAg was assessed. Scale bar: 10 μm. The fluorescence intensity of SHBsAg and HBcAg were analyzed using ImageJ software. The intracellular distribution of SHBsAg and HBcAg was analyzed by determining their intensity profiles along the white arrows using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. (E and F) Huh7 cells transiently transfected with pSM2 were cotransfected with si DNM2 or si CLTC , and the CCDC88A expression plasmid or empty vector (E), or si RAB5A and the CCDC88A expression plasmid or empty vector (F), harvested after 72 h. The levels of HBsAg and HBeAg in the supernatants and that in the intracellular were quantified. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Anti-DNM2 Antibody , Cell Signaling Technology, 2342.

Techniques: Blocking Assay, Transfection, Expressing, Fluorescence, Software, Plasmid Preparation

Journal: Autophagy

Article Title: CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

doi: 10.1080/15548627.2021.1934271

Figure Lengend Snippet: Inhibition of DNM2 by dynasore suppresses HBV replication. (A) Huh7 cells were transfected with pSM2, and treated with dynasore (20 μM) and harvested after 48 h. RAB5A expression was evaluated. Scale bar: 10 μm. The fluorescence intensity of RAB5A was analyzed using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. (B) Huh7 cells transfected with pSM2 and HepG2.2.15 cells were treated with dynasore (5, 10, 15, and 20 μM), and harvested after 72 h. The levels of HBsAg and HBeAg in the supernatants were quantified. (C) Huh7 cells transiently transfected with pSM2 or (D) HepG2.2.15 cells were treated with dynasore (20 μM) and harvested after 72 h. The expression of SHBsAg and HBcAg, the encapsidated HBV RIs, the levels of intracellular HBV DNA, and the HBV RNA levels were measured. (E) Huh7 cells transfected with pSM2 and HepG2.2.15 cells were treated with dynasore (20 μM), and harvested after 48 h. The distribution of SHBsAg and HBcAg was assessed. Scale bar: 10 μm. The fluorescence intensity of SHBsAg and HBcAg were analyzed using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. (F) Huh7 cells were cotransfected with pSM2 and the CCDC88A expression plasmid or empty vector, and treated with dynasore (20 μM), and harvested after 72 h. The expression of SHBsAg and HBcAg, and the levels of HBsAg and HBeAg in the supernatants were analyzed. * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Anti-DNM2 Antibody , Cell Signaling Technology, 2342.

Techniques: Inhibition, Transfection, Expressing, Fluorescence, Software, Plasmid Preparation

Journal: Autophagy

Article Title: CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

doi: 10.1080/15548627.2021.1934271

Figure Lengend Snippet: CCDC88A or DNM2 silencing reduces colocalization of SHBsAg with early endosomes and prevents core particles from entering MVBs. Huh7 cells were cotransfected with pSM2, with (A) or without (B) RAB5A-EGFP plasmids for 48 h. (C) Huh7 cells were cotransfected with pSM2 and si CCDC88A or si DNM2 for 48 h. The colocalization of SHBsAg with RAB5A was imaged. Scale bar: 10 μm. (D) Huh7 cells were cotransfected with pSM2 and si CCDC88A or si DNM2 for 48 h. (E) Huh7 cells were transfected with HBV deletion mutant plasmids, including LHBsAg − , MHBsAg − , SHBsAg − , L/MHBsAg − and AllHBsAg − plasmids for 48 h. The colocalization of HBcAg and CD63 was measured. Scale bar: 10 μm. The fluorescence intensity of target proteins, and the colocalization between target proteins or with organelle marker proteins were analyzed using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. (F) Huh7 cells were transfected with the HBV wild-type (WT) and HBV deletion mutant plasmids, including LHBsAg − , MHBsAg − , SHBsAg − , L/MHBsAg − and AllHBsAg − plasmids, for 72 h. The HBV gene expression and replication were analyzed. (G) Huh7 cells were transfected with the HBV WT plasmid or cotransfected with the HBV deletion mutant plasmid SHBsAg − , and HA-SHBsAg, HA-pol or empty vector for 48 h. The expression of HBcAg and SHBsAg or HA was detected. Scale bar: 10 μm. *, # p < 0.05; **, ## p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Anti-DNM2 Antibody , Cell Signaling Technology, 2342.

Techniques: Transfection, Mutagenesis, Fluorescence, Marker, Software, Gene Expression, Plasmid Preparation, Expressing

Journal: Autophagy

Article Title: CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

doi: 10.1080/15548627.2021.1934271

Figure Lengend Snippet: CCDC88A or DNM2 silencing causes SHBsAg accumulation on the ER lumen, thereby triggering ER stress. Huh7 cells were cotransfected with pSM2 combined without (A) or with (B) DsRed-ER plasmid, and si CCDC88A or si DNM2 for 48 h. The colocalization of SHBsAg and ER was imaged. Scale bar: 10 μm. The colocalization between SHBsAg and ER was analyzed using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. (C) Huh7 cells were cotransfected with pSM2 or empty plasmid pUC19, and si CCDC88A or si DNM2 for 48 h. HepG2.2.15 cells were transfected with si CCDC88A or si DNM2 (D) or treated with dynasore (E) for 48 h. The expression levels of HSPA5, EIF2A, p-EIF2A, ERN1, p-ERN1 and ATF6 were measured.

Article Snippet: Anti-DNM2 Antibody , Cell Signaling Technology, 2342.

Techniques: Plasmid Preparation, Software, Transfection, Expressing

Journal: Autophagy

Article Title: CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

doi: 10.1080/15548627.2021.1934271

Figure Lengend Snippet: CCDC88A or DNM2 silencing leads to increased autophagic flux and HBV antigens degradation. (A) Huh7 cells were cotransfected with pSM2 and si CCDC88A or si DNM2 for 48 h. The colocalization of SHBsAg and LC3 was imaged. Scale bar: 10 μm. (B) HepG2.2.15 cells were transfected with si CCDC88A , and then treated with 10 μM CQ, and harvested after 72 h. The expression of SHBsAg, HBcAg, LC3 and SQSTM1 (upper) and the levels of HBsAg and HBeAg in the supernatants (below) were quantified. (C) Huh7 cells were cotransfected with pSM2 or empty plasmid pUC19 and si CCDC88A or si DNM2 , and harvested after 48 h and then incubated with DQ-BSA Red for 30 min. The fluorescent signal of DQ-BSA Red produced by autolysosomal proteolysis was evaluated. Scale bar: 10 μm. (D) Huh7 cells were cotransfected with the mCherry-GFP-LC3 plasmid and si CCDC88A or si DNM2 , and harvested after 48 h. The mCherry and GFP signals were examined by confocal microscopy. Scale bar: 10 μm. (E and F) Huh7 cells were cotransfected with pSM2 and si CCDC88A or si DNM2 . The fluorescence intensity of LysoTracker Red (E) or AO (F) were analyzed. Scale bar: 10 μm. The fluorescence intensity of target proteins, and the colocalization between target proteins or with organelle marker proteins were analyzed using ImageJ software. The results presented in the graphs were calculated from at least 5 cells. *, # p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant.

Article Snippet: Anti-DNM2 Antibody , Cell Signaling Technology, 2342.

Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Produced, Confocal Microscopy, Fluorescence, Marker, Software

Journal: Autophagy

Article Title: CCDC88A/GIV promotes HBV replication and progeny secretion via enhancing endosomal trafficking and blocking autophagic degradation

doi: 10.1080/15548627.2021.1934271

Figure Lengend Snippet: List of antibodies

Article Snippet: Anti-DNM2 Antibody , Cell Signaling Technology, 2342.

Techniques:

Journal: The Journal of Neuroscience

Article Title: Interaction with the Unfolded Protein Response Reveals a Role for Stargazin in Biosynthetic AMPA Receptor Transport

doi: 10.1523/JNEUROSCI.3568-04.2005

Figure Lengend Snippet: Control experiments for the immunocytochemical and biochemical assays used in this study to distinguish surface and intracellular protein pools. A-C, Representative images showing immunostaining with anti-HA before (Surface) and after (Internal) permeabilization with Triton X-100. A, COS7 cells were transfected with HA-tagged wild-type GABAB receptor (GABABR) GB1 subunit, known to be retained in the ER. B, COS7 cells were transfected with the HA-tagged AA/ASRR mutant of the GB1 subunit, known to express heavily on the cell surface. C, Wild-type GB1 subunit remains undetectable on the cell surface after coexpression of stargazin. Scale bar, 25 μm. D, COS7 cells were transfected with an HA-tagged version of the cytosolic protein dynamin I (K44E mutant) and subjected to the immunoprecipitation assay described in Materials and Methods. After SDS-PAGE of 1% of the internal fraction and 50% of the surface fraction, the blot was probed with anti-dynamin. Dynamin was only detected in the intracellular fraction, excluding contamination of the surface fraction by intracellular protein.

Article Snippet: As a control, COS7 cells were transfected with an HA-tagged version of the cytosolic protein dynamin I (K44E mutant) and subjected to the immunoprecipitation and SDS-PAGE assay described above, followed by immunoblotting with anti-dynamin (H-300; Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Control, Immunostaining, Transfection, Mutagenesis, Immunoprecipitation, SDS Page

Journal: eLife

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

doi: 10.7554/eLife.14530

Figure Lengend Snippet: ( A ) Glutamate release by synaptosomes, monitored by enzymatic conversion of glutamate by glutamate dehydrogenase (GluDH) (see Materials and methods). The functionality of isolated synaptosomes was also assessed by immunoblot detection of phosphorylated Ser603 of synapsin1 and Ser774 of dynamin1 ( B and C ). ( A ) Synaptosomes were preincubated in buffer containing either Ca 2+ (final concentration 1.3 mM) or EGTA (final concentration 0.5 mM), followed by sequential addition of GluDH (200 U) and KCl (K + ) for depolarization (final concentration 50 mM) or not stimulated (no K + was added). Data represented as mean of three replicates, with the bars indicating the range of values. ( B and C ) Immunoblot analyses of phosphorylation changes in synapsin1 (Ser603) and dynamin1 (Ser774). Synaptosomes monitored and treated 2 min after addition of KCl as described in ( A ) were analyzed by immunoblotting using phosphospecific antibodies. ( B ) Representative immunoblots. To ensure equal loading, all samples were also blotted using antibodies insensitive to phosphorylation change, and tubulin as a loading control. ( C ) Quantification of the blot signals obtained from three independent experiments shown as the mean of the three replicates, with the error bars indicating the range of values. DOI: http://dx.doi.org/10.7554/eLife.14530.003

Article Snippet: The following primary antibodies were used: synapsin1, dynamin1-3 and α-tubulin (Synaptic Systems, Göttingen, Germany), phospho S774-dynamin1, phospho S603-synapsin1 (Rockland, Pottstow, PA).

Techniques: Isolation, Western Blot, Concentration Assay, Phospho-proteomics, Control

Journal: eLife

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

doi: 10.7554/eLife.14530

Figure Lengend Snippet: Partial spectra of mass spectrometric survey scans in MS1 (precursor spectra) containing phosphopeptides of synapsin Ser603 ( A ) and dynamin S774 ( B ) in light (K + , Ca 2+ ) and heavy (K + , EGTA) isotopic pattern that is used to calculated the relative abundances. MS/MS spectra resulting from HCD fragmentation of phosphopeptides containing synapsin Ser603 m/z 538.7478 2+ ( C ) and dynamin Ser774 m/z 703.2123 2+ ( D ), respectively, showing the phosphopeptides _QAS(ph)QAGPGPR_ and _RS(ph)PTS(ph)SPTPQR. Both of these phosphopeptides were eluted in the first fraction of SCX. MaxQuant Viewer (version 1.5.0.25) was used to illustrate the spectra. DOI: http://dx.doi.org/10.7554/eLife.14530.009

Article Snippet: The following primary antibodies were used: synapsin1, dynamin1-3 and α-tubulin (Synaptic Systems, Göttingen, Germany), phospho S774-dynamin1, phospho S603-synapsin1 (Rockland, Pottstow, PA).

Techniques: Tandem Mass Spectroscopy

Journal: eLife

Article Title: Analysis of protein phosphorylation in nerve terminal reveals extensive changes in active zone proteins upon exocytosis

doi: 10.7554/eLife.14530

Figure Lengend Snippet: Colors indicate phosphosites that are upregulated (red), downregulated (green), or remain unchanged (black) in depolarization with Ca 2+ versus depolarization without Ca 2+ comparison (K + , Ca 2+ versus K + , EGTA). The underlined phosphosites represent sites previously reported to be physiologically relevant (for references see Supplementary file 3, available at Dryad ). Unmarked phosphosites represent sites previously reported only by proteomic discovery-mode mass spectrometry, defined as HTP in the publicly available PhosphoSitePlus database. Phosphosites marked with a star represent phosphosites reported here for the first time. For comparison, phosphosites found in the well-studied proteins dynamin1 and synapsin1 are shown. DOI: http://dx.doi.org/10.7554/eLife.14530.013

Article Snippet: The following primary antibodies were used: synapsin1, dynamin1-3 and α-tubulin (Synaptic Systems, Göttingen, Germany), phospho S774-dynamin1, phospho S603-synapsin1 (Rockland, Pottstow, PA).

Techniques: Comparison, Mass Spectrometry