Journal: PLoS ONE

Article Title: Quantitative Kinetic Study of the Actin-Bundling Protein L-Plastin and of Its Impact on Actin Turn-Over

doi: 10.1371/journal.pone.0009210

Figure Lengend Snippet: (A). Novel PKC isozymes regulate PMA-induced L-plastin phosphorylation in MCF-7 cells. MCF-7 cells were pretreated for 3 hours with 5 µM of GF109203X (specific for α, β1, δ, ε, and ζ) or 0.5 µM of Gö6976 (specific for α and β1) and then treated for 1 hour with or without 1 µM PMA at 37°C. Total cell extracts (50 µg) were analyzed by immunoblotting using anti-Ser5-P L-plastin (upper panel) or anti-GAPDH (lower panel) antibodies. (B). PMA-induced actin cytoskeleton remodeling and L-plastin translocation involves novel PKC isozymes. MCF-7 cells were pretreated for 3 hours with 5 µM GF109203X or 0.5 µM Gö6976, then treated for 1 hour with 1 µM PMA at 37°C. Cells were then fixed and processed for immunofluorescence. Labeled cells were analyzed with an epifluorescence microscope after staining with an anti-L-plastin antibody and Alexa 488-conjugated phalloidin. Scale bar, 10 µm. (C). SiRNA knock-down of PKC-δ decreased PMA-induced L-plastin phosphorylation in MCF-7. MCF-7 cells were transfected with either PKC-δ or PKC-ε siRNAs as well as with negative control siRNA (Ctrl) for 48 hours and then treated with PMA as indicated. Total cell extracts (50 µg) were analyzed by immunoblotting using antibodies specific for Ser5 phosphorylated L-plastin, PKC-ε, PKC-δ and total L-plastin. GAPDH was used to monitor equal protein loading.

Article Snippet: Rabbit polyclonal anti-PKC-δ and rabbit monoclonal anti-PKC-ε were from Cell Signaling Technology, Inc. (Danvers, MA).

Techniques: Western Blot, Translocation Assay, Immunofluorescence, Labeling, Microscopy, Staining, Transfection, Negative Control

Journal: Drug Design, Development and Therapy

Article Title: Involvement of microRNA-146a in diabetic peripheral neuropathy through the regulation of inflammation

doi: 10.2147/DDDT.S157109

Figure Lengend Snippet: Expression of NF-κB in the sciatic nerve of each group. ( A ) Western blot analysis showed the expression of NF-κB p65 and p50 in the sciatic nerve of each group. ( B ) The relative expression of NF-κB p65 and p50 normalized to β-actin ( # P <0.01). Abbreviations: NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; T2DM, type 2 diabetes mellitus; DPN, diabetic peripheral neuropathy.

Article Snippet: The primary antibodies used were a monoclonal rabbit p65 antibody (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), p50 antibody (1:1,000 dilution; Cell Signaling Technology) and an anti-β-actin primary antibody (1:5,000 dilution; Proteintech Group Inc., Chicago, IL, USA).

Techniques: Expressing, Western Blot

Journal: Drug Design, Development and Therapy

Article Title: Involvement of microRNA-146a in diabetic peripheral neuropathy through the regulation of inflammation

doi: 10.2147/DDDT.S157109

Figure Lengend Snippet: The association between miR-146a, cytokines and NF-κB. Correlation between ( A ) miR-146a and IL-1β ( r 2 =0.7683, P <0.01), ( B ) TNF-α ( r 2 =0.8491, P <0.01), ( C ) NF-κB p50 ( r 2 =0.8898, P <0.01) and ( D ) NF-κB p65 ( r 2 =0.8945, P <0.01). Abbreviations: miR-146a, microRNA-146a; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; IL-1β, interleukin 1 beta; TNF-α, tumor necrosis factor alpha.

Article Snippet: The primary antibodies used were a monoclonal rabbit p65 antibody (1:1,000 dilution; Cell Signaling Technology, Danvers, MA, USA), p50 antibody (1:1,000 dilution; Cell Signaling Technology) and an anti-β-actin primary antibody (1:5,000 dilution; Proteintech Group Inc., Chicago, IL, USA).

Techniques:

Journal: PLoS ONE

Article Title: Fragile X Related Protein 1 Clusters with Ribosomes and Messenger RNAs at a Subset of Dendritic Spines in the Mouse Hippocampus

doi: 10.1371/journal.pone.0026120

Figure Lengend Snippet: A. Hippocampal lysates were prepared from mice at different developmental stages (P2 = postnatal day 2) and analyzed for FXR1P, FXR2P, FMRP, L7 ribosomal protein and GAPDH. Isoforms of FXR1P (a.b,c,d), FXR2P, FMRP and ribosomal protein L7 were all highly expressed during early postnatal development in the hippocampus. B. FXR1P (isoforms c, d) expression across postnatal development was quantified and normalized against GAPDH expression. FXR1P levels decreased relative to GAPDH. C. We immunostained cryostat sections prepared from a P14 mouse with #ML13 and imaged the hippocampus at 10X (left panel). We found that FXR1P was highly expressed in neurons at P14. A 60X image of pyramidal neurons in area CA1 of the hippocampus (box in 10X image) showing FXR1P expression in the cell body and proximal dendrites of CA1 neurons (right panel). Arrow points to a proximal dendrite found in the plane of the image. Scale bar = 60 µm (low magnification) and 10 µm (high magnification)

Article Snippet: Other antibodies used included mouse monoclonal antibodies against FMRP (mAb1C3; ), FXR1P (mAb3FX; ), FXR2P (mAbA42, Abcam), myc (Santa Cruz; 9E10), MAP-2 (Sigma-Aldrich; HM-2)and GAPDH (Abcam; ab9484), human anti-ribosomal P antibodies (Immunovision), a rabbit anti-ribosomal large protein L7 (Cell Signaling), a rabbit monoclonal antibody against S6 (Cell Signaling; 5G10) and a goat polyclonal antibody against TIA-1 (Santa Cruz; sc-1751).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Fragile X Related Protein 1 Clusters with Ribosomes and Messenger RNAs at a Subset of Dendritic Spines in the Mouse Hippocampus

doi: 10.1371/journal.pone.0026120

Figure Lengend Snippet: Aliquots of native polyribosomes and EDTA treated polyribosomes were loaded onto linear 15–45% (w/w) sucrose gradients and centrifuged for 2 hr at 34 000 rpm at 4°C in a Beckman SW40 rotor. Each collected fraction was assayed for the presence of FXR1P and L7 ribosomal protein. Fractions from the top to the bottom of the gradient are shown from left to right and the position of the 80 S ribosome monomer is indicated. SS, LS: ribosomal small and large subunits, respectively.

Article Snippet: Other antibodies used included mouse monoclonal antibodies against FMRP (mAb1C3; ), FXR1P (mAb3FX; ), FXR2P (mAbA42, Abcam), myc (Santa Cruz; 9E10), MAP-2 (Sigma-Aldrich; HM-2)and GAPDH (Abcam; ab9484), human anti-ribosomal P antibodies (Immunovision), a rabbit anti-ribosomal large protein L7 (Cell Signaling), a rabbit monoclonal antibody against S6 (Cell Signaling; 5G10) and a goat polyclonal antibody against TIA-1 (Santa Cruz; sc-1751).

Techniques:

Journal: Molecular cell

Article Title: Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression

doi: 10.1016/j.molcel.2018.10.034

Figure Lengend Snippet: (A and B) Western blot analysis of reciprocally co-immunoprecipitated endogenous RB and p65 proteins in PC-3 cells.

Article Snippet: Rabbit monoclonal anti-p65 , Cell Signaling Technology , Cat# 8242; RRID: AB_10859369.

Techniques: Western Blot, Immunoprecipitation

Journal: Molecular cell

Article Title: Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression

doi: 10.1016/j.molcel.2018.10.034

Figure Lengend Snippet: (A) (Top) Schematic diagram depicting CDK4/CYCLIN D phosphorylation sites (P letter) of RB-N. (Bottom) Western blot analysis of p65 proteins in PC-3 whole-cell lysate (WCL) pulled down by GST-RB. Asterisks indicate proteins at the expected molecular weight.

Article Snippet: Rabbit monoclonal anti-p65 , Cell Signaling Technology , Cat# 8242; RRID: AB_10859369.

Techniques: Western Blot, Molecular Weight

Journal: Molecular cell

Article Title: Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression

doi: 10.1016/j.molcel.2018.10.034

Figure Lengend Snippet: (A) CYCLIN D/CDK4/6-mediated RB S249/T252 phosphorylation promotes specific RB-p65 interaction, thereby triggering inhibition of expression of NF-κB target genes such as PD-L1 and cancer immunity.

Article Snippet: Rabbit monoclonal anti-p65 , Cell Signaling Technology , Cat# 8242; RRID: AB_10859369.

Techniques: Inhibition, Expressing

Journal: Molecular cell

Article Title: Phosphorylated RB Promotes Cancer Immunity by Inhibiting NF-κB Activation and PD-L1 Expression

doi: 10.1016/j.molcel.2018.10.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-p65 , Cell Signaling Technology , Cat# 8242; RRID: AB_10859369.

Techniques: Recombinant, Mutagenesis, Kinase Assay, Sequencing, CRAfT Assay, Software