Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Subcellular colocalization of 24K and miRNA biogenesis proteins. ( A ) (i) Colocalization of 24K:mRFP with CFP:AtDCL1 in nuclear aggregates. ( B ) (i) Colocalization of 24K:mRFP with AtHYL1:YFP in nuclear aggregates. ( C ) (i) Colocalization of 24K:mRFP with AtSE:YFP in nuclear aggregates. (ii) The mRFP control does not colocalize in nuclear aggregates with any of the tested proteins. ( D ) Western blot analyses of extracts from N. benthamiana leaves in colocalization assays at 3 dpa. Anti-RFP (@RFP) or anti-GFP (@GFP) monoclonal antibodies were used for 24K or biogenesis proteins, respectively. Coomassie blue stain is shown as loading control. The numbers below correspond to normalized band density. Ratios between AtDCL and AtDCL1/24K (240 kDa); AtHYL1 and AtHYL1/24K (75 kDa); AtSE and AtSE/24K (110 kDa) are shown under the lines. Scale bar: 10 µm.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: Control, Western Blot, Bioprocessing, Staining

Journal: Microbiology Spectrum

Article Title: Citrus psorosis virus 24K protein inhibits the processing of miRNA precursors by interacting with components of the biogenesis machinery

doi: 10.1128/spectrum.03513-23

Figure Lengend Snippet: Interaction between 24K protein and miRNA biogenesis proteins. ( A ) In vivo analysis of BiFC assays in epidermal cells of N. benthamiana plants. The merge panels show positive interaction between N-mCitrine:24K and C-mCitrine:AtHYL1 (i) and N-mCitrine:24K and C-mCitrine:AtSE (ii) and no interaction between N-mCitrine:24K with C-mCitrine:AtDCL1 (iii). (iv). Negative control: N-mCitrine:24K + C-mCitrine (empty vector). Chloroplasts were marked in blue in the case of the negative interactions. In all cases, Fib:mRFP was used as an expression control of infiltrated samples. Scale bar: 10 µm. ( B ) Co-IP assay between 24K:mRFP and AtHYL1. INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins. (−) corresponds to the negative control using beads without @HYL1 antibody. (+) corresponds to the immunoprecipitated samples with @HYL1 attached to the beads. The anti-RFP (@RFP) and anti-HYL1 (@HYL1) monoclonal antibodies were used in each case to develop the blots. The presence of both proteins in the IP fraction indicates a positive interaction. ( C ) Co-IP assay between the 24K:mRFP and AtSE:YFP protein. The INPUT and UNBOUND controls correspond to input and output fractions without binding to bead system, respectively. The IP fraction corresponds to immunoprecipitated proteins with @GFP attached to the beads. Free mRFP was used as a negative control. The anti-RFP (@RFP) and anti-GFP (@GFP) antibodies were used in each case to develop the blots. The presence of both proteins in IP fraction indicates a positive interaction.

Article Snippet: GFP (or its variants, CFP or YFP) and RFP fusion proteins were detected with anti-GFP (3H9) monoclonal antibody (Chromotek, Germany) and anti-RFP (6G6) monoclonal antibody (Chromotek, Germany), respectively.

Techniques: In Vivo, Negative Control, Plasmid Preparation, Expressing, Control, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Bioprocessing

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Sumoylation of La increases STAT3 mRNA binding by La. (A) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in GFP-LaWT (Wt)- and GFP-LaSD (K41/200R)-expressing cells. The values are normalized against GAPDH mRNA levels (n = 3). (B) RT-PCR on RNA samples prepared from RIP experiments using HEK293 cells stably overexpressing GFP-LaWT (Wt) or GFP-LaSD (K41/200R). Significantly less STAT3 mRNA was associated with GFP-LaSD than with GFP-LaWT. The asterisks indicate a significant difference (P < 0.01), as determined by Student's t test (n = 3). The data represent means and SD of the results of independent experiments.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Stable Transfection

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Sumoylation of La promotes cell proliferation via a STAT3-mediated mechanism. (A) Representative immunoblot of STAT3 in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. GAPDH was used as a loading control. (B) RT-qPCR analysis showing no significant difference in STAT3 mRNA levels in HEK293 cells transduced with shC, sh3, and sh5 lentiviral constructs. The values are normalized against GAPDH mRNA levels (n = 3). (C) Representative immunoblot showing STAT3 protein levels in GFP-LaWT- and GFP-LaSD-expressing cells. GAPDH was used as a loading control. (D) Densitometry analysis showing significantly lower STAT3 protein expression in GFP-LaSD-expressing cells than in GFP-LaWT-expressing cells. The values are normalized against GAPDH protein levels (n = 3). (E) Representative fluorescence images showing transient transfection of control (GFP) and RFP-STAT3 in cells transduced with lentiviral constructs expressing shC or sh5. The transfection efficiency was ∼30%. (F) Overexpression of STAT3 (RFP-STAT3) restored the numbers of La-depleted (sh5) cells (n = 4; *, P = 0.0358). (G) Representative fluorescence images showing transient transfection of control and RFP-STAT3 in GFP-, GFP-LaWT-, and GFP-LaSD-expressing cells. The transfection efficiency was ∼30%. (H) Overexpression of STAT3 (RFP-STAT3) restored GFP-LaSD cell numbers (n = 3; *, P = 0.015). The asterisks indicate significant differences (P < 0.05), as determined by Student's t test. NS, not significant. The data represent means and SD of the results of independent experiments.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Western Blot, Transduction, Construct, Quantitative RT-PCR, Expressing, Fluorescence, Transfection, Over Expression

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Global translation is not impaired in GFP-LaWT or GFP-LaSD cells, and sumoylation of La has a minor impact on STAT3 mRNA translation in HEK293 cells. (A and B) Autoradiography (A) and corresponding Coomassie-stained gel (B) of [35S]methionine-labeled total proteins of GFP-LaWT- and GFP-LaSD-expressing cells (30 min and 60 min). (C) Overlay of polyribosome fractionation profiles of two gradients loaded with extracts from GFP-LaWT (Wt-I/II) cells and two gradients loaded with extracts from GFP-LaSD (SD-I/II) cells. (D) STAT3 mRNA distribution in polyribosomal gradients from GFP-LaWT or GFP-LaSD cells as analyzed by RT-qPCR. (E) GADPH mRNA distribution in polyribosomal gradients from GFP-LaWT or GFP-LaSD cells as analyzed by RT-qPCR. The results are representative of three independent experiments.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Autoradiography, Staining, Labeling, Expressing, Fractionation, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: SUMO Modification of the RNA-Binding Protein La Regulates Cell Proliferation and STAT3 Protein Stability

doi: 10.1128/MCB.00129-17

Figure Lengend Snippet: Sumoylation of La promotes STAT3 protein stability. (A) GFP-LaWT- and GFP-LaSD-expressing cells were treated with CHX (20 μM) for the indicated times and analyzed for STAT3 expression by immunoblot analysis. GAPDH protein levels were analyzed as a loading control. (B) Quantification of immunoblots revealed that STAT3 stability was reduced in GFP-LaSD cells compared to GFP-LaWT (n = 3; *, P = 0.022 at 6 h and P = 0.023 at 12 h). The asterisks indicate significant differences (P < 0.05), as determined by Student's t test. (C) GFP-LaWT- and GFP-LaSD-expressing cells were treated with CHX (20 μM) and the proteasome inhibitor MG132 (10 μg/ml) for the indicated times and analyzed for STAT3 expression by immunoblot analysis. GAPDH protein levels were analyzed as a loading control. (D) Quantification of immunoblots revealed no significant difference in STAT3 stability in GFP-LaSD and GFP-LaWT cells (n = 3; P = 0.24 at 6 h; P = 0.51 at 12 h). (E) Representative immunoblot showing global ubiquitination in GFP-LaWT and GFP-LaSD cells treated or not treated with the proteasome inhibitor MG132 (10 μg/ml). GAPDH protein levels were analyzed as a loading control. (F) Representative immunoblot showing ubiquitination of STAT3 in GFP-LaSD cells in the absence or presence of MG132. GFP-LaWT- and GFP-LaSD-expressing cells were cotransfected with Flag-tagged STAT3 (STAT3-Flag) and HA-tagged ubiquitin (UB-HA). After 24 h, the cells were treated or not treated with MG132 (10 μg/ml) and subjected to immunoprecipitation applying a Flag-specific antibody. The immunoblots were analyzed with HA-specific (detection of ubiquitin) or Flag-specific (detection of STAT3) antibody. Protein levels in the extracts (Input) used for IP (bottom) were also assessed.

Article Snippet: For STAT3 overexpression, the control (pEGFP-C1) or the RFP-STAT3 (Sino Biological Inc.) plasmid was transiently transfected into La-depleted or GFP-La SD -expressing cells using FuGene HD transfection reagent (Promega).

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: iScience

Article Title: Phosphatidylserine synthase plays an essential role in glia and affects development, as well as the maintenance of neuronal function

doi: 10.1016/j.isci.2021.102899

Figure Lengend Snippet:

Article Snippet: Mouse anti-GFP (G1-S, 1:25, Developmental Studies Hybridoma Bank (DSHB)), rabbit anti-GFP (1:25, Abcam), mouse anti-Repo (8D12, 1:20, DSHB), mouse anti-Elav (9F8A9, 1:40, DSHB), and mouse anti-CSP (ab49, 1/20, DSHB) were used as primary antibodies.

Techniques: Virus, Recombinant, SYBR Green Assay, Software, Real-time Polymerase Chain Reaction