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: Protein Science : A Publication of the Protein Society

Article Title: Insect cell expression and purification of recombinant SARS‐COV ‐2 spike proteins that demonstrate ACE2 binding

doi: 10.1002/pro.4300

Figure Lengend Snippet: Functional S‐Ecto‐eGFP binding is reduced by ACE2 receptor blockade. Calu3 cells were incubated with 40 μg/ml α‐ACE2 or goat IgG antibody (IgG) for 45 min prior to incubation with the S1‐ectodomain‐eGFP protein. Data are represented as the fold increase of GFP MFI (median fluorescent intensity) compared to GFP‐His tag control (mean ± SEM ). N = 4 independent experiments. * p < .05

Article Snippet: For staining, all test samples were incubated with 50 μg/ml S‐Ecto‐eGFP or control GFP‐His tag protein (Sino Biological) and Zombie NIR Fixable Viability dye (BioLegend) in PBS/2% FBS for 30 min, rocking at RT.

Techniques: Functional Assay, Binding Assay, Incubation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Insect cell expression and purification of recombinant SARS‐COV ‐2 spike proteins that demonstrate ACE2 binding

doi: 10.1002/pro.4300

Figure Lengend Snippet: Reduced functional S‐Ecto‐eGFP binding to surface ACE2 in BET‐inhibitor treated Calu3 cells. Calu3 cells were treated with BET inhibitors (JQ1, RVX‐208; RVX) or control DMSO vehicle (VEH) for 24 hr prior to incubation with the S1‐ectodomain‐eGFP protein. Data are represented as the fold increase of GFP MFI (median fluorescent intensity) compared to GFP‐His tag control (mean ± SEM ). N = 3–4 independent experiments. * p < .05, ** p < .001

Article Snippet: For staining, all test samples were incubated with 50 μg/ml S‐Ecto‐eGFP or control GFP‐His tag protein (Sino Biological) and Zombie NIR Fixable Viability dye (BioLegend) in PBS/2% FBS for 30 min, rocking at RT.

Techniques: Functional Assay, Binding Assay, Incubation

Journal: Journal of Virology

Article Title: A Lysine Residue Essential for Geminivirus Replication Also Controls Nuclear Localization of the Tomato Yellow Leaf Curl Virus Rep Protein

doi: 10.1128/JVI.01910-18

Figure Lengend Snippet: Nuclear localization of Rep from two TYLCV strains is controlled by the conserved lysines. (A) Subcellular localization of RFP-tagged RepTYLCV (TYLCV-Alb13) variants in N. benthamiana upon transient expression with Agrobacterium. Arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. Scale bars represent 5 μm. (B) Immunoblot of the Rep-RFP fusion proteins upon transient expression in N. benthamiana. Proteins were detected with an anti-RFP antibody. To demonstrate equal protein loading, the membranes were stained with Ponceau S. (C) Micrographs of epidermal leaf cells transiently expressing RepTYLCV K-to-A triple mutant-RFP and stained with ER-Tracker Green. From the left in order: RFP channel, ER-Tracker, merge of RFP and ER-Tracker, bright field, and graph representing the normalized fluorescence intensity of RFP and ER-Tracker (GFP) along the lines in the micrographs. Note that the two signals are shifted and do not overlap, indicating that they localize in different positions. Scale bars represent 50 μm. (D) Subcellular localization of RFP-tagged Rep TYLCV-Almeria WT and K-to-A variants in N. benthamiana. For each sample, one representative epidermal cell is shown with a 4× zoom of its nucleus; arrowheads indicate fluorescence in the cytoplasm, and asterisks mark nonfluorescent or weakly fluorescent nuclei. The scale bars represent 5 μm. (E) Box plot showing the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel D; a total of 16 cells were analyzed. (F) Box plot depicting the RFP fluorescence intensity ratio in the cytoplasm versus nucleus for the images shown in panel A; a total of 8 cells per sample were analyzed. The statistical analysis used is described in the legend to Fig. 2.

Article Snippet: Immunodetection of the proteins was performed according to standard protocols using anti-RFP antibody (Chromotek 6G6; 1:1,000) to detect the Rep-RFP fusion proteins and antihemagglutinin (anti-HA) antibody (Roche 3F10; 1:2,000) for Rep-SCFP C fusions as primary antibodies and anti-rat (Pierce 31470; 1:10,000) or anti-mouse (Pierce 31430; 1:10,000) as secondary antibodies.

Techniques: Expressing, Fluorescence, Western Blot, Staining, Mutagenesis

Journal: iScience

Article Title: Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation

doi: 10.1016/j.isci.2022.104196

Figure Lengend Snippet:

Article Snippet: C57BL/6J mice were tail-vein injected with pCMV3- CDH1 (Sino Biological, mouse/MG50671-CF, ) or EGFP (Sino Biological, Aequorea Victoria/AG13105-CF) using PEI Max transfection reagent (PEI, Polysciences) according to a method modified from .

Techniques: Recombinant, Chromatin Immunoprecipitation, AST Assay, Immunoprecipitation, shRNA, Luciferase, Software

Journal: Nature Communications

Article Title: Open-channel microfluidics via resonant wireless power transfer

doi: 10.1038/s41467-022-29405-2

Figure Lengend Snippet: a A green fluorescent protein (GFP) reservoir drop (green) and Alexa Fluor-594 reservoir drop (red) were mixed using 25 parallel channels after applying 3.5 V RMS . Conglomerates of GFP were extruded from the GFP reservoir into individual OMEF channels and were labeled red by the Alexa dye after mixing using amine chemistry. b Alexa fluorescence intensity in the GFP reservoir is plotted versus time (red) and normalized to its source-reservoir fluorescence at the same time point. Similarly, GFP fluorescence inside the Alexa reservoir is also plotted versus time (green). An exchange in reservoir material is observed for both solutions. Source data are provided as a Source Data file. c Schematic demonstrating wireless inductive coupling of magnetic fields to actuate liquid. d Demonstration of wireless smartphone actuation of PBS using an NFC signal. Images are from before (left) and after (right) smartphone-powered actuation was applied, where the solution was pulled an additional ~200 µm beyond the capillary action while increasing the channel width throughout its entire length. The electrode edge is outlined in white for reference. Scale bar is 25 µm. Below is an example of a resonant wireless power-transfer circuit using a primary inductor ( L P ), capacitor ( C P ), and resistor ( R P ) (LCR) circuit to transfer power to a secondary LCR with an inductance ( L S ), resistance ( R S ), and circuit capacitance of our device, C DEP .

Article Snippet: Protein mixing and labeling experiments utilized green fluorescent protein (GFP) modified with a polyhistidine tag (λ ex : 487 nm, λ em : 508 nm, GFPSpark, Sino Biological).

Techniques: Labeling, Fluorescence