Structured Review

Millipore anti β actin monoclonal antibody
The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A , vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) ( left panel ) or IRF-3(5A) ( right panel ) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μ m ) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and <t>anti-β-actin</t> antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) ( top ) panel is presented in supplemental Fig. S1 , to confirm IRF-3 can be visualized in lanes 1 2 -18. B , vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK ( Z-VAD ) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A , with the exception that the cells were treated with Z-VAD-FMK (10 μ m ) in place of MG132 ( left panel ) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μ m ), or both Z-VAD-FMK (10 μ m ) and MG132 (10 μ m ) ( right panel ). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP ( Un-Cl , 116 kDa) and one cleaved fragment ( Cleaved , 83 kDa). This experiment is representative of more than three performed independently.
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Images

1) Product Images from "Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein *"

Article Title: Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.033290

The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A , vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) ( left panel ) or IRF-3(5A) ( right panel ) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μ m ) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and anti-β-actin antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) ( top ) panel is presented in supplemental Fig. S1 , to confirm IRF-3 can be visualized in lanes 1 2 -18. B , vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK ( Z-VAD ) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A , with the exception that the cells were treated with Z-VAD-FMK (10 μ m ) in place of MG132 ( left panel ) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μ m ), or both Z-VAD-FMK (10 μ m ) and MG132 (10 μ m ) ( right panel ). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP ( Un-Cl , 116 kDa) and one cleaved fragment ( Cleaved , 83 kDa). This experiment is representative of more than three performed independently.
Figure Legend Snippet: The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A , vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) ( left panel ) or IRF-3(5A) ( right panel ) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μ m ) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and anti-β-actin antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) ( top ) panel is presented in supplemental Fig. S1 , to confirm IRF-3 can be visualized in lanes 1 2 -18. B , vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK ( Z-VAD ) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A , with the exception that the cells were treated with Z-VAD-FMK (10 μ m ) in place of MG132 ( left panel ) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μ m ), or both Z-VAD-FMK (10 μ m ) and MG132 (10 μ m ) ( right panel ). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP ( Un-Cl , 116 kDa) and one cleaved fragment ( Cleaved , 83 kDa). This experiment is representative of more than three performed independently.

Techniques Used: Transfection, Expressing, FLAG-tag, Plasmid Preparation, Negative Control, SDS Page, Activity Assay

2) Product Images from "NF-κB Activation Promotes Alphavirus Replication in Mature Neurons"

Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

Journal: Journal of Virology

doi: 10.1128/JVI.01071-19

SINV infection of differentiated AP-7 cells induced prolonged canonical NF-κB activation. dAP-7 cells were infected with SINV or mock infected (with medium) at 39°C. (A) Immunoblot analysis of cell lysates for canonical NF-κB activation using antibodies against phosphorylated p65 (S536), IκBα, phosphorylated IKKα/β (S176/S177), and total IKKβ; viral protein production using antibodies against SINV nonstructural protein 2 (nsP2); and β-actin. hpi, hours postinfection. (B) Densitometric analysis of the levels of phosphorylated p65 and IκBα normalized to those of β-actin. Data are presented as the mean ± SD from three independent experiments. **, P
Figure Legend Snippet: SINV infection of differentiated AP-7 cells induced prolonged canonical NF-κB activation. dAP-7 cells were infected with SINV or mock infected (with medium) at 39°C. (A) Immunoblot analysis of cell lysates for canonical NF-κB activation using antibodies against phosphorylated p65 (S536), IκBα, phosphorylated IKKα/β (S176/S177), and total IKKβ; viral protein production using antibodies against SINV nonstructural protein 2 (nsP2); and β-actin. hpi, hours postinfection. (B) Densitometric analysis of the levels of phosphorylated p65 and IκBα normalized to those of β-actin. Data are presented as the mean ± SD from three independent experiments. **, P

Techniques Used: Infection, Activation Assay

TPCA-1 treatment inhibited canonical NF-κB activation in differentiated AP-7 cells. dAP-7 cells were pretreated with the NF-κB inhibitor TPCA-1 or DMSO for 1 h at the indicated concentrations and stimulated with recombinant rat TNF-α (10 μg/ml) for 30 min at 39°C. (A) Protein lysates were analyzed by immunoblotting for phosphorylated p65 (S536) and β-actin. (B) Cells were fixed and analyzed by immunocytochemistry for p65 localization. Images were taken at a magnification of ×40 and are representative of those from two independent experiments.
Figure Legend Snippet: TPCA-1 treatment inhibited canonical NF-κB activation in differentiated AP-7 cells. dAP-7 cells were pretreated with the NF-κB inhibitor TPCA-1 or DMSO for 1 h at the indicated concentrations and stimulated with recombinant rat TNF-α (10 μg/ml) for 30 min at 39°C. (A) Protein lysates were analyzed by immunoblotting for phosphorylated p65 (S536) and β-actin. (B) Cells were fixed and analyzed by immunocytochemistry for p65 localization. Images were taken at a magnification of ×40 and are representative of those from two independent experiments.

Techniques Used: Activation Assay, Recombinant, Immunocytochemistry

Inhibition of NF-κB activation had little effect on SINV replication in cycling AP-7 cells or mouse embryonic fibroblasts. cAP-7 cells were infected with SINV and treated with TPCA-1 (10 μM) or DMSO at 33°C. (A) Protein lysates were assessed by immunoblotting for phosphorylated p65 (S536), IκBα, SINV structural proteins, and β-actin. (B) Supernatants from cAP-7 cells were assessed for infectious virus by plaque assay in BHK-21 cells. (C) Wild-type (WT) or IKKβ-deficient mouse embryonic fibroblasts (MEFs) were infected with SINV (MOI, 1) at 37°C. Supernatants were assessed for infectious virus by plaque assay in BHK-21 cells. Data are presented as the mean ± SD from three independent experiments.
Figure Legend Snippet: Inhibition of NF-κB activation had little effect on SINV replication in cycling AP-7 cells or mouse embryonic fibroblasts. cAP-7 cells were infected with SINV and treated with TPCA-1 (10 μM) or DMSO at 33°C. (A) Protein lysates were assessed by immunoblotting for phosphorylated p65 (S536), IκBα, SINV structural proteins, and β-actin. (B) Supernatants from cAP-7 cells were assessed for infectious virus by plaque assay in BHK-21 cells. (C) Wild-type (WT) or IKKβ-deficient mouse embryonic fibroblasts (MEFs) were infected with SINV (MOI, 1) at 37°C. Supernatants were assessed for infectious virus by plaque assay in BHK-21 cells. Data are presented as the mean ± SD from three independent experiments.

Techniques Used: Inhibition, Activation Assay, Infection, Plaque Assay

The absence of IKKβ decreased SINV replication in differentiated AP-7 cells more than in cycling AP-7 cells. (A) Immunoblot analysis of wild-type (WT) dAP-7 cells and IKKβ-deficient dAP-7 cells infected with SINV at 39°C. Protein lysates were probed for phosphorylated IKKα/β (S176/S177), total IKKβ, phosphorylated p65 (S536), total p65, SINV structural proteins, SINV nsP2, and β-actin. (B, C) Infectious virus production by SINV-infected WT and IKKβ-deficient differentiated AP-7 cells (B) and cycling AP-7 cells (C), as measured by plaque assay in BHK-21 cells. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P
Figure Legend Snippet: The absence of IKKβ decreased SINV replication in differentiated AP-7 cells more than in cycling AP-7 cells. (A) Immunoblot analysis of wild-type (WT) dAP-7 cells and IKKβ-deficient dAP-7 cells infected with SINV at 39°C. Protein lysates were probed for phosphorylated IKKα/β (S176/S177), total IKKβ, phosphorylated p65 (S536), total p65, SINV structural proteins, SINV nsP2, and β-actin. (B, C) Infectious virus production by SINV-infected WT and IKKβ-deficient differentiated AP-7 cells (B) and cycling AP-7 cells (C), as measured by plaque assay in BHK-21 cells. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

Techniques Used: Infection, Plaque Assay

NF-κB inhibition reduced SINV structural protein production in differentiated AP-7 cells. dAP-7 cells were infected with SINV and treated with either TPCA-1 (10 μM) or DMSO at 39°C. (A) Whole-cell lysates were probed by immunoblotting with antibodies against phosphorylated p65, IκBα, SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and β-actin. (B to D) Densitometric analysis of phosphorylated p65 (B), SINV capsid (C), and SINV nsP2 (D) normalized to β-actin. Data are presented as the mean ± SD from three independent experiments. *, P
Figure Legend Snippet: NF-κB inhibition reduced SINV structural protein production in differentiated AP-7 cells. dAP-7 cells were infected with SINV and treated with either TPCA-1 (10 μM) or DMSO at 39°C. (A) Whole-cell lysates were probed by immunoblotting with antibodies against phosphorylated p65, IκBα, SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and β-actin. (B to D) Densitometric analysis of phosphorylated p65 (B), SINV capsid (C), and SINV nsP2 (D) normalized to β-actin. Data are presented as the mean ± SD from three independent experiments. *, P

Techniques Used: Inhibition, Infection

SINV replication and induction of NF-κB activation in differentiated and cycling AP-7 cells. cAP-7 and dAP-7 cells were infected with SINV at 33°C and 39°C, respectively. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Reverse-phase protein array (RPPA) analysis of phosphorylated p65 (p-p65; S536) normalized to total p65 and IκBα normalized to β-actin in SINV-infected cAP-7 and dAP-7 cells. Values indicate the fold increase relative to the level in matched mock-infected samples. Data are presented as the mean ± SD for triplicate samples. **, P
Figure Legend Snippet: SINV replication and induction of NF-κB activation in differentiated and cycling AP-7 cells. cAP-7 and dAP-7 cells were infected with SINV at 33°C and 39°C, respectively. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Reverse-phase protein array (RPPA) analysis of phosphorylated p65 (p-p65; S536) normalized to total p65 and IκBα normalized to β-actin in SINV-infected cAP-7 and dAP-7 cells. Values indicate the fold increase relative to the level in matched mock-infected samples. Data are presented as the mean ± SD for triplicate samples. **, P

Techniques Used: Activation Assay, Infection, Plaque Assay, Protein Array

Effect of PKR inhibition results on SINV replication in mature neurons. dAP-7 cells were infected with SINV and treated with the PKR inhibitor C16 (1 μM) or DMSO at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Immunoblot analysis for phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), total eIF2α, and β-actin. (C) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P
Figure Legend Snippet: Effect of PKR inhibition results on SINV replication in mature neurons. dAP-7 cells were infected with SINV and treated with the PKR inhibitor C16 (1 μM) or DMSO at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Immunoblot analysis for phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), total eIF2α, and β-actin. (C) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

Techniques Used: Inhibition, Infection, Plaque Assay

Effect of PKR absence on SINV replication in mature neurons. WT and PKR-deficient dAP-7 cells were infected with SINV at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Cell viability relative to day 0 (D0) cell counts, as determined by trypan blue exclusion. (C) Immunoblot analysis for PKR, phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and total eIF2α. (D) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P
Figure Legend Snippet: Effect of PKR absence on SINV replication in mature neurons. WT and PKR-deficient dAP-7 cells were infected with SINV at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Cell viability relative to day 0 (D0) cell counts, as determined by trypan blue exclusion. (C) Immunoblot analysis for PKR, phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and total eIF2α. (D) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

Techniques Used: Infection, Plaque Assay

3) Product Images from "Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice"

Article Title: Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0141258

Protein expression level of CAV2. (A) Western blotting revealed an increase in CAV2 level in Cx26 f/f P0Cre mice at postnatal day 21. (B) CAV level was normalized to that of β-actin and is expressed as relative to the amount present in each littermate control. Values represent the mean ± SE (n = 6 for control, n = 5 for Cx26 f/f P0Cre). **P = 2.5×10 −3 for CAV2, Student’s t -test.
Figure Legend Snippet: Protein expression level of CAV2. (A) Western blotting revealed an increase in CAV2 level in Cx26 f/f P0Cre mice at postnatal day 21. (B) CAV level was normalized to that of β-actin and is expressed as relative to the amount present in each littermate control. Values represent the mean ± SE (n = 6 for control, n = 5 for Cx26 f/f P0Cre). **P = 2.5×10 −3 for CAV2, Student’s t -test.

Techniques Used: Expressing, Western Blot, Mouse Assay

4) Product Images from "A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters"

Article Title: A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters

Journal: Biological Procedures Online

doi: 10.1251/bpo147

Neuropeptide expression in uninduced and induced MSCs. A. Uninduced (D0) and induced (D12) MSCs were co-labeled with PE-anti-SP and FITC-anti-SV2 or FITC-anti-synaptophysin (Syn), and then counterlabeled with nucleus-specific DAPI. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification. B. Media from uninduced (D0) and induced (D6 and D12) MSCs was immunoprecipitated with anti-VIP and anti-CGRP and then analyzed by western blot. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-CGRP and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification. D. Whole cell extracts from D0, D6 and D12 cells were prepared and analyzed by western blots with anti-Leu-Enkephalin. Normalizations were performed with anti-β-actin. Representative blots are shown for three different experiments. Band quantification was performed as in B *p
Figure Legend Snippet: Neuropeptide expression in uninduced and induced MSCs. A. Uninduced (D0) and induced (D12) MSCs were co-labeled with PE-anti-SP and FITC-anti-SV2 or FITC-anti-synaptophysin (Syn), and then counterlabeled with nucleus-specific DAPI. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification. B. Media from uninduced (D0) and induced (D6 and D12) MSCs was immunoprecipitated with anti-VIP and anti-CGRP and then analyzed by western blot. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-CGRP and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification. D. Whole cell extracts from D0, D6 and D12 cells were prepared and analyzed by western blots with anti-Leu-Enkephalin. Normalizations were performed with anti-β-actin. Representative blots are shown for three different experiments. Band quantification was performed as in B *p

Techniques Used: Expressing, Labeling, Immunoprecipitation, Western Blot, Fluorescence

Expression of GABA and glutamate receptors in uninduced (D0) and induced (D12) MSCs. A. Total RNA from D0 and D12 cells was studied for expression of the GABAA A receptor β1 subunit and NMDA1 receptor by RT-PCR. Normalizations were performed with oligonucleotides specific for GAPDH. Representative gel is shown for three different experiments. B. Whole cell extracts from D0 and D12 cells were prepared and analyzed by western blots with anti-GABAAR-β1 and anti-NMDAR1. Normalizations were performed with anti-β-actin. Representative blots are shown for three different experiments.
Figure Legend Snippet: Expression of GABA and glutamate receptors in uninduced (D0) and induced (D12) MSCs. A. Total RNA from D0 and D12 cells was studied for expression of the GABAA A receptor β1 subunit and NMDA1 receptor by RT-PCR. Normalizations were performed with oligonucleotides specific for GAPDH. Representative gel is shown for three different experiments. B. Whole cell extracts from D0 and D12 cells were prepared and analyzed by western blots with anti-GABAAR-β1 and anti-NMDAR1. Normalizations were performed with anti-β-actin. Representative blots are shown for three different experiments.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

Expression of CNS neurotransmitters in uninduced and induced MSCs. A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p
Figure Legend Snippet: Expression of CNS neurotransmitters in uninduced and induced MSCs. A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p

Techniques Used: Expressing, Western Blot, Positive Control, Labeling, Fluorescence

5) Product Images from "SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy"

Article Title: SMAD4 gene mutation renders pancreatic cancer resistance to radiotherapy through promotion of autophagy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-17-3435

SMAD4 depletion induced high level of autophagy in response to ionizing radiation. ( A-C ) Panc-1 and MIA PaCa-2 were transfected with scrambled siRNA or SMAD4 -specific siRNA for 48 h and then were treated with or without IR (6 Gy). Cell lysates were harvested at the indicated time points after IR. ( A ) Immunoblot of LC3 isoforms were measured using antibody against LC3. Quantification of the LC3-II/LC3-I ratio in Panc-1 cells ( B ) and MIA PaCa-2 cells ( C ). The protein level of the β-actin was used as the internal standard. Shown are the averages of triplicate experiments. ( D ) Representative confocal images of endogenous LC3 staining in Panc-1 transfected with scramble or SMAD4 -specific siRNA for 48 h, and treated with or without IR (6 Gy). Cells were stained with antibody to LC3 (red) and DAPI (blue). Scale bars, 10 μm. ( E ) Quantification of LC3 puncta in Panc-1 cell lines.
Figure Legend Snippet: SMAD4 depletion induced high level of autophagy in response to ionizing radiation. ( A-C ) Panc-1 and MIA PaCa-2 were transfected with scrambled siRNA or SMAD4 -specific siRNA for 48 h and then were treated with or without IR (6 Gy). Cell lysates were harvested at the indicated time points after IR. ( A ) Immunoblot of LC3 isoforms were measured using antibody against LC3. Quantification of the LC3-II/LC3-I ratio in Panc-1 cells ( B ) and MIA PaCa-2 cells ( C ). The protein level of the β-actin was used as the internal standard. Shown are the averages of triplicate experiments. ( D ) Representative confocal images of endogenous LC3 staining in Panc-1 transfected with scramble or SMAD4 -specific siRNA for 48 h, and treated with or without IR (6 Gy). Cells were stained with antibody to LC3 (red) and DAPI (blue). Scale bars, 10 μm. ( E ) Quantification of LC3 puncta in Panc-1 cell lines.

Techniques Used: Transfection, Staining

6) Product Images from "Cladribine in combination with entinostat synergistically elicits anti-proliferative/anti-survival effects on multiple myeloma cells"

Article Title: Cladribine in combination with entinostat synergistically elicits anti-proliferative/anti-survival effects on multiple myeloma cells

Journal: Cell Cycle

doi: 10.1080/15384101.2018.1464849

The combinations of cladribine and entinostat potently influence expression of the molecular markers involving in DNA damage response. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to western blot analyses with specific antibody directed against P-H2A.X, P-Chk1, Chk1, P-Chk2, Chk2, or β-actin. (a) RPMI8226; (b) U226; (c) MM1.R.
Figure Legend Snippet: The combinations of cladribine and entinostat potently influence expression of the molecular markers involving in DNA damage response. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to western blot analyses with specific antibody directed against P-H2A.X, P-Chk1, Chk1, P-Chk2, Chk2, or β-actin. (a) RPMI8226; (b) U226; (c) MM1.R.

Techniques Used: Expressing, Cell Culture, Western Blot

The combinations of cladribine and entinostat markedly alter expression of several key molecular markers critical for G1-S transition. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to western blot analyses with specific antibody directed against Cyclin D1, E2F-1, p21 waf−1 , p27 kip−1 , or β-actin. The densitometry analyses of p21 waf-1 and Cyclin D1 signals are shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0. (a) RPMI8226; (b) U226; (c) MM1.R.
Figure Legend Snippet: The combinations of cladribine and entinostat markedly alter expression of several key molecular markers critical for G1-S transition. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of entinostat (Ent), cladribine (Clad) alone or their combinations for 48 hrs. Cells were collected and subjected to western blot analyses with specific antibody directed against Cyclin D1, E2F-1, p21 waf−1 , p27 kip−1 , or β-actin. The densitometry analyses of p21 waf-1 and Cyclin D1 signals are shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0. (a) RPMI8226; (b) U226; (c) MM1.R.

Techniques Used: Expressing, Cell Culture, Western Blot

Combination of cladribine and entinostat significantly promotes MM cells undergoing apoptosis. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of either entinostat (Ent) or cladribine (Clad) alone, or their combinations for 48 hrs. Cells were collected and subjected to apoptotic ELISA or western blot analyses with specific antibody directed against PARP (F-PARP, full length PARP; C-PARP, cleaved PARP), caspase-3 (F-Casp-3, full length caspase-3; C-Casp-3, cleaved caspase-3), caspase-8 (F-Casp-8, full length caspase-8; C-Casp-8, cleaved caspase-8), caspase-9 (F-Casp-9, full length caspase-9; C-Casp-9, cleaved caspase-9), or β-actin. Bars, SD. (a) RPMI8226; (b) U226; (c) MM1.R.
Figure Legend Snippet: Combination of cladribine and entinostat significantly promotes MM cells undergoing apoptosis. MM cells were cultured with RPMI1640 (2.5% FBS) in the absence or presence of either entinostat (Ent) or cladribine (Clad) alone, or their combinations for 48 hrs. Cells were collected and subjected to apoptotic ELISA or western blot analyses with specific antibody directed against PARP (F-PARP, full length PARP; C-PARP, cleaved PARP), caspase-3 (F-Casp-3, full length caspase-3; C-Casp-3, cleaved caspase-3), caspase-8 (F-Casp-8, full length caspase-8; C-Casp-8, cleaved caspase-8), caspase-9 (F-Casp-9, full length caspase-9; C-Casp-9, cleaved caspase-9), or β-actin. Bars, SD. (a) RPMI8226; (b) U226; (c) MM1.R.

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

7) Product Images from "Carfilzomib and ONX 0912 Inhibit Cell Survival and Tumor Growth of Head and Neck Cancer and Their Activities are Enhanced by Suppression of Mcl-1 or Autophagy"

Article Title: Carfilzomib and ONX 0912 Inhibit Cell Survival and Tumor Growth of Head and Neck Cancer and Their Activities are Enhanced by Suppression of Mcl-1 or Autophagy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

doi: 10.1158/1078-0432.CCR-12-1213

Carfilzomib and ONX 0912 induce prosurvival autophagy in HNSCC cells. A, HNSCC cells were treated for 24 hours with 100 nmol/L carfilzomib or ONX 0912, or with 0.1% DMSO. Immunoblots were probed for Beclin-1, Atg5/12 conjugate, LC3-II, or β-actin.
Figure Legend Snippet: Carfilzomib and ONX 0912 induce prosurvival autophagy in HNSCC cells. A, HNSCC cells were treated for 24 hours with 100 nmol/L carfilzomib or ONX 0912, or with 0.1% DMSO. Immunoblots were probed for Beclin-1, Atg5/12 conjugate, LC3-II, or β-actin.

Techniques Used: Western Blot

Related Articles

Blocking Assay:

Article Title: Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice
Article Snippet: The proteins were resolved by SDS-PAGE using mini-PROTEAN TGX gradient gels (4–20% polyacrylamide; Bio-Rad Laboratories, Inc.) and then transferred to a polyvinylidene difluoride membrane (Amersham Hybond-P; GE Healthcare). .. After blocking, each membrane was processed through sequential incubations with anti-CAV2 (1:500, Sigma Aldrich) and monoclonal anti-β-actin (1:1500; Sigma Aldrich) with horseradish peroxidase—conjugated anti-rabbit or anti-mouse IgG (1:40,000; GE Healthcare) as the secondary antibody. .. Amersham ELC Prime Western Blotting Detection Reagent (GE Healthcare) was then used for visualization, and the signal was observed by Image Quant LAS 4000 (Fujifilm).

other:

Article Title: Carfilzomib and ONX 0912 Inhibit Cell Survival and Tumor Growth of Head and Neck Cancer and Their Activities are Enhanced by Suppression of Mcl-1 or Autophagy
Article Snippet: Immunoblotting utilized antibodies directed against Bcl-XL (cat. #sc-8392), Mcl-1 (sc-819), Bik (sc-10770), Bax (sc-493), Beclin-1 (sc-11427), total PERK (sc-13073), ATF4 (sc-200)(Santa Cruz Biotechnology, Inc.), Bcl-2 (M0887)(Dako Corp.), Bim (AAP-330) and caspase-3 (AAP-113)(Assay Designs), Bak (3814), PARP (9542) and phospho-eIF2α (3597; Ser51)(Cell Signaling Technology), LC3 (NB100-2220) and Atg5/12 (NB110-53818)(Novus Biologicals, Inc.), total eIF2α (606702) and phospho-PERK (649402; Ser713)(BioLegend), and β-actin (A5441)(Sigma).

Western Blot:

Article Title: Cladribine in combination with entinostat synergistically elicits anti-proliferative/anti-survival effects on multiple myeloma cells
Article Snippet: .. The sources of antibodies for western blot assays were as follows: caspase-3 rabbit mAb (8G10), caspase-8 (1C12) mouse mAb, caspase-9 (Asp353) rabbit mAb, PARP rabbit mAb, P-Histone H2A.X (Ser139) rabbit antibody, Acetyl-Histone H3 (Lys9), Histone H3, P-CHK1 (Ser345) (133D3) rabbit mAb, CHK1 rabbit antibody, P-CHK2 (Thr68) rabbit polyclonal antibody, CHK2 rabbit polyclonal antibody and p21Waf1/Cip1 (12D1) rabbit mAb (Cell Signaling Technology, Inc., Beverly, MA); Cyclin D1 rabbit mAb, E2F-1 mouse mAb (KH95), p27 (F-8) mouse mAb (Santa Cruz Biotechnology Inc., Santa Cruz, CA); β-actin mouse mAb (clone AC-75) (Sigma Co.). ..

Article Title: Direct RNA sequencing reveals m6A modifications on adenovirus RNA are necessary for efficient splicing
Article Snippet: .. Antibodies, immunoblotting, and immunofluorescenceThe following primary antibodies were used for cellular proteins: METTL3 (Novus Biologicals H00056339, WB: 1:400, IF: 1:50), METTL14 (Sigma-Aldrich HPA038002, WB: 1:5000, IF: 1:100), WTAP (Proteintech 60188, WB: 1:400, IF: 1:100), YTHDC1 (Abcam ab122340, WB: 1:2000, IF: 1:100), YTHDF1 (Abcam ab99080, WB: 1:500, IF: 1:100), YTHDF2 (Proteintech 24744-1-AP, WB: 1;1000, IF: 1:100), YTHDF3 (Sigma-Aldrich SAB2102735, WB: 1:500), FTO (Abcam ab92821, WB: 1:300, IF: 1:100), ALKBH5 (Sigma-Aldrich SAB1407587, WB: 1:250, IF: 1:100), β-Actin (Sigma-Aldrich A5441-100UL, WB 1:5000), GAPDH (GeneTex 41577, WB:1:20,000), and RNA Pol II p-Ser2 (Abcam ab5095, IF: 1:400). .. Primary anti-Flag tag antibody was obtained from Sigma-Aldrich (F7425-.2MG, WB 1:2000).

Article Title: Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein *
Article Snippet: .. Antibodies for Western Blot and Immunoprecipitation Primary antibodies were used at the following dilutions and incubation conditions: anti-Pro-caspase-3 polyclonal antibody (Cell Signaling Technologies) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-poly(ADP-ribose) polymerase monoclonal antibody (Sigma-Aldrich) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-phospho-IRF-3 (Ser396) polyclonal antibody (Upstate) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-FLAG® M2 monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-XpressTM monoclonal antibody (Invitrogen) diluted 1:5,000 and incubated for 1 h at 4 °C; anti-β-actin monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-IRF-3 monoclonal antibody (Abcam) diluted 1:100 and incubated for 2 h at 4 °C; and for denaturing gels IRF-3 was detected with anti-IRF-3(FL-425) polyclonal antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated 1 h at 4 °C. ..

Immunoprecipitation:

Article Title: Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein *
Article Snippet: .. Antibodies for Western Blot and Immunoprecipitation Primary antibodies were used at the following dilutions and incubation conditions: anti-Pro-caspase-3 polyclonal antibody (Cell Signaling Technologies) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-poly(ADP-ribose) polymerase monoclonal antibody (Sigma-Aldrich) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-phospho-IRF-3 (Ser396) polyclonal antibody (Upstate) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-FLAG® M2 monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-XpressTM monoclonal antibody (Invitrogen) diluted 1:5,000 and incubated for 1 h at 4 °C; anti-β-actin monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-IRF-3 monoclonal antibody (Abcam) diluted 1:100 and incubated for 2 h at 4 °C; and for denaturing gels IRF-3 was detected with anti-IRF-3(FL-425) polyclonal antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated 1 h at 4 °C. ..

Incubation:

Article Title: Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein *
Article Snippet: .. Antibodies for Western Blot and Immunoprecipitation Primary antibodies were used at the following dilutions and incubation conditions: anti-Pro-caspase-3 polyclonal antibody (Cell Signaling Technologies) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-poly(ADP-ribose) polymerase monoclonal antibody (Sigma-Aldrich) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-phospho-IRF-3 (Ser396) polyclonal antibody (Upstate) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-FLAG® M2 monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-XpressTM monoclonal antibody (Invitrogen) diluted 1:5,000 and incubated for 1 h at 4 °C; anti-β-actin monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-IRF-3 monoclonal antibody (Abcam) diluted 1:100 and incubated for 2 h at 4 °C; and for denaturing gels IRF-3 was detected with anti-IRF-3(FL-425) polyclonal antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated 1 h at 4 °C. ..

Immunodetection:

Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons
Article Snippet: Ten micrograms of total protein was separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a nitrocellulose membrane (Bio-Rad), and blocked in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) and 5% milk. .. Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore). .. The secondary antibodies horseradish peroxidase-conjugated goat anti-rabbit IgG (1:1,000; Cell Signaling Technology) or sheep anti-mouse IgG (1:1,000; GE Healthcare) were diluted 1:1,000 in 5% milk.

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  • 99
    Millipore rhodamine phalloidin
    HT1080 fibroblast adhesion and spreading on collagen-mimetic protein-coated wells. (A) Adhesion numbers on protein coats after a 3-h incubation period. (B) Average cell spreading (area) at 3 h. (C) Rhodamine <t>phalloidin</t> and SYBR Green-stained HT1080 fibroblasts. n =4 wells per protein, 3 images per well for a total of 12 images per sample; mean±standard deviation displayed; ◯‡* indicate statistically significant differences between respective samples p
    Rhodamine Phalloidin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti β actin monoclonal antibody
    The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A , vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) ( left panel ) or IRF-3(5A) ( right panel ) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μ m ) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and <t>anti-β-actin</t> antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) ( top ) panel is presented in supplemental Fig. S1 , to confirm IRF-3 can be visualized in lanes 1 2 -18. B , vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK ( Z-VAD ) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A , with the exception that the cells were treated with Z-VAD-FMK (10 μ m ) in place of MG132 ( left panel ) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μ m ), or both Z-VAD-FMK (10 μ m ) and MG132 (10 μ m ) ( right panel ). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP ( Un-Cl , 116 kDa) and one cleaved fragment ( Cleaved , 83 kDa). This experiment is representative of more than three performed independently.
    Anti β Actin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HT1080 fibroblast adhesion and spreading on collagen-mimetic protein-coated wells. (A) Adhesion numbers on protein coats after a 3-h incubation period. (B) Average cell spreading (area) at 3 h. (C) Rhodamine phalloidin and SYBR Green-stained HT1080 fibroblasts. n =4 wells per protein, 3 images per well for a total of 12 images per sample; mean±standard deviation displayed; ◯‡* indicate statistically significant differences between respective samples p

    Journal: Advances in Wound Care

    Article Title: Chronic Wound Dressings Based on Collagen-Mimetic Proteins

    doi: 10.1089/wound.2014.0614

    Figure Lengend Snippet: HT1080 fibroblast adhesion and spreading on collagen-mimetic protein-coated wells. (A) Adhesion numbers on protein coats after a 3-h incubation period. (B) Average cell spreading (area) at 3 h. (C) Rhodamine phalloidin and SYBR Green-stained HT1080 fibroblasts. n =4 wells per protein, 3 images per well for a total of 12 images per sample; mean±standard deviation displayed; ◯‡* indicate statistically significant differences between respective samples p

    Article Snippet: Cells were stained with rhodamine phalloidin (actin/cytoplasm; Sigma-Aldrich) and SYBR Green (DNA/nucleus) and imaged (three images per sample, four samples per protein) on a DeltaVision Elite microscope (GE Healthcare).

    Techniques: Incubation, SYBR Green Assay, Staining, Standard Deviation

    The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A , vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) ( left panel ) or IRF-3(5A) ( right panel ) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μ m ) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and anti-β-actin antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) ( top ) panel is presented in supplemental Fig. S1 , to confirm IRF-3 can be visualized in lanes 1 2 -18. B , vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK ( Z-VAD ) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A , with the exception that the cells were treated with Z-VAD-FMK (10 μ m ) in place of MG132 ( left panel ) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μ m ), or both Z-VAD-FMK (10 μ m ) and MG132 (10 μ m ) ( right panel ). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP ( Un-Cl , 116 kDa) and one cleaved fragment ( Cleaved , 83 kDa). This experiment is representative of more than three performed independently.

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of Caspase-mediated Decay of Interferon Regulatory Factor-3, Exploited by a Kaposi Sarcoma-associated Herpesvirus Immunoregulatory Protein *

    doi: 10.1074/jbc.M109.033290

    Figure Lengend Snippet: The accelerated decay of activated wild-type IRF-3 by vIRF-2 is independent of the proteasome, but inhibited by a general caspase inhibitor. A , vIRF-2-accelerated decay of wild-type IRF-3 is independent of the proteasome. HEK293 cells were transiently co-transfected with expression vectors for either FLAG epitope-tagged wild type IRF-3 (IRF-3WT) ( left panel ) or IRF-3(5A) ( right panel ) and either Xpress epitope-tagged vIRF-2, or the empty parental plasmid backbone, pcDNA4. Twenty-four hours later, cells were treated for 30 min with either DMSO (as the vehicle negative control) or MG132 (10 μ m ) and then transfected with poly(I:C) (10 μg/ml). Lysates were prepared at various times thereafter, and protein samples were separated by SDS-PAGE and immunoblotted with anti-FLAG (to detect IRF-3), anti-Xpress (to detect vIRF-2), and anti-β-actin antibodies. This experiment is representative of more than three performed independently. A longer exposure of the anti-FLAG (IRF-3) ( top ) panel is presented in supplemental Fig. S1 , to confirm IRF-3 can be visualized in lanes 1 2 -18. B , vIRF-2-accelerated decay of wild-type IRF-3 is repressed by Z-VAD-FMK ( Z-VAD ) treatment, which inhibits caspase activity. This experiment was repeated essentially as described for A , with the exception that the cells were treated with Z-VAD-FMK (10 μ m ) in place of MG132 ( left panel ) and the IRF-3(5A) expression plasmid was not used. Alternatively, cells were treated with DMSO, MG132 (10 μ m ), or both Z-VAD-FMK (10 μ m ) and MG132 (10 μ m ) ( right panel ). Immunoblotting was performed with anti-FLAG (IRF-3), anti-Xpress (vIRF-2), anti-PARP, and anti-β-actin antibodies. The anti-PARP antibody recognizes uncleaved PARP ( Un-Cl , 116 kDa) and one cleaved fragment ( Cleaved , 83 kDa). This experiment is representative of more than three performed independently.

    Article Snippet: Antibodies for Western Blot and Immunoprecipitation Primary antibodies were used at the following dilutions and incubation conditions: anti-Pro-caspase-3 polyclonal antibody (Cell Signaling Technologies) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-poly(ADP-ribose) polymerase monoclonal antibody (Sigma-Aldrich) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-phospho-IRF-3 (Ser396) polyclonal antibody (Upstate) diluted 1:5,000 and incubated for 18 h at 4 °C; anti-FLAG® M2 monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-XpressTM monoclonal antibody (Invitrogen) diluted 1:5,000 and incubated for 1 h at 4 °C; anti-β-actin monoclonal antibody (Sigma-Aldrich) diluted 1:50,000 and incubated for 1 h at 4 °C; anti-IRF-3 monoclonal antibody (Abcam) diluted 1:100 and incubated for 2 h at 4 °C; and for denaturing gels IRF-3 was detected with anti-IRF-3(FL-425) polyclonal antibody (Santa Cruz Biotechnology) diluted 1:1000 and incubated 1 h at 4 °C.

    Techniques: Transfection, Expressing, FLAG-tag, Plasmid Preparation, Negative Control, SDS Page, Activity Assay

    SINV infection of differentiated AP-7 cells induced prolonged canonical NF-κB activation. dAP-7 cells were infected with SINV or mock infected (with medium) at 39°C. (A) Immunoblot analysis of cell lysates for canonical NF-κB activation using antibodies against phosphorylated p65 (S536), IκBα, phosphorylated IKKα/β (S176/S177), and total IKKβ; viral protein production using antibodies against SINV nonstructural protein 2 (nsP2); and β-actin. hpi, hours postinfection. (B) Densitometric analysis of the levels of phosphorylated p65 and IκBα normalized to those of β-actin. Data are presented as the mean ± SD from three independent experiments. **, P

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: SINV infection of differentiated AP-7 cells induced prolonged canonical NF-κB activation. dAP-7 cells were infected with SINV or mock infected (with medium) at 39°C. (A) Immunoblot analysis of cell lysates for canonical NF-κB activation using antibodies against phosphorylated p65 (S536), IκBα, phosphorylated IKKα/β (S176/S177), and total IKKβ; viral protein production using antibodies against SINV nonstructural protein 2 (nsP2); and β-actin. hpi, hours postinfection. (B) Densitometric analysis of the levels of phosphorylated p65 and IκBα normalized to those of β-actin. Data are presented as the mean ± SD from three independent experiments. **, P

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Infection, Activation Assay

    TPCA-1 treatment inhibited canonical NF-κB activation in differentiated AP-7 cells. dAP-7 cells were pretreated with the NF-κB inhibitor TPCA-1 or DMSO for 1 h at the indicated concentrations and stimulated with recombinant rat TNF-α (10 μg/ml) for 30 min at 39°C. (A) Protein lysates were analyzed by immunoblotting for phosphorylated p65 (S536) and β-actin. (B) Cells were fixed and analyzed by immunocytochemistry for p65 localization. Images were taken at a magnification of ×40 and are representative of those from two independent experiments.

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: TPCA-1 treatment inhibited canonical NF-κB activation in differentiated AP-7 cells. dAP-7 cells were pretreated with the NF-κB inhibitor TPCA-1 or DMSO for 1 h at the indicated concentrations and stimulated with recombinant rat TNF-α (10 μg/ml) for 30 min at 39°C. (A) Protein lysates were analyzed by immunoblotting for phosphorylated p65 (S536) and β-actin. (B) Cells were fixed and analyzed by immunocytochemistry for p65 localization. Images were taken at a magnification of ×40 and are representative of those from two independent experiments.

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Activation Assay, Recombinant, Immunocytochemistry

    Inhibition of NF-κB activation had little effect on SINV replication in cycling AP-7 cells or mouse embryonic fibroblasts. cAP-7 cells were infected with SINV and treated with TPCA-1 (10 μM) or DMSO at 33°C. (A) Protein lysates were assessed by immunoblotting for phosphorylated p65 (S536), IκBα, SINV structural proteins, and β-actin. (B) Supernatants from cAP-7 cells were assessed for infectious virus by plaque assay in BHK-21 cells. (C) Wild-type (WT) or IKKβ-deficient mouse embryonic fibroblasts (MEFs) were infected with SINV (MOI, 1) at 37°C. Supernatants were assessed for infectious virus by plaque assay in BHK-21 cells. Data are presented as the mean ± SD from three independent experiments.

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: Inhibition of NF-κB activation had little effect on SINV replication in cycling AP-7 cells or mouse embryonic fibroblasts. cAP-7 cells were infected with SINV and treated with TPCA-1 (10 μM) or DMSO at 33°C. (A) Protein lysates were assessed by immunoblotting for phosphorylated p65 (S536), IκBα, SINV structural proteins, and β-actin. (B) Supernatants from cAP-7 cells were assessed for infectious virus by plaque assay in BHK-21 cells. (C) Wild-type (WT) or IKKβ-deficient mouse embryonic fibroblasts (MEFs) were infected with SINV (MOI, 1) at 37°C. Supernatants were assessed for infectious virus by plaque assay in BHK-21 cells. Data are presented as the mean ± SD from three independent experiments.

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Inhibition, Activation Assay, Infection, Plaque Assay

    The absence of IKKβ decreased SINV replication in differentiated AP-7 cells more than in cycling AP-7 cells. (A) Immunoblot analysis of wild-type (WT) dAP-7 cells and IKKβ-deficient dAP-7 cells infected with SINV at 39°C. Protein lysates were probed for phosphorylated IKKα/β (S176/S177), total IKKβ, phosphorylated p65 (S536), total p65, SINV structural proteins, SINV nsP2, and β-actin. (B, C) Infectious virus production by SINV-infected WT and IKKβ-deficient differentiated AP-7 cells (B) and cycling AP-7 cells (C), as measured by plaque assay in BHK-21 cells. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: The absence of IKKβ decreased SINV replication in differentiated AP-7 cells more than in cycling AP-7 cells. (A) Immunoblot analysis of wild-type (WT) dAP-7 cells and IKKβ-deficient dAP-7 cells infected with SINV at 39°C. Protein lysates were probed for phosphorylated IKKα/β (S176/S177), total IKKβ, phosphorylated p65 (S536), total p65, SINV structural proteins, SINV nsP2, and β-actin. (B, C) Infectious virus production by SINV-infected WT and IKKβ-deficient differentiated AP-7 cells (B) and cycling AP-7 cells (C), as measured by plaque assay in BHK-21 cells. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Infection, Plaque Assay

    NF-κB inhibition reduced SINV structural protein production in differentiated AP-7 cells. dAP-7 cells were infected with SINV and treated with either TPCA-1 (10 μM) or DMSO at 39°C. (A) Whole-cell lysates were probed by immunoblotting with antibodies against phosphorylated p65, IκBα, SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and β-actin. (B to D) Densitometric analysis of phosphorylated p65 (B), SINV capsid (C), and SINV nsP2 (D) normalized to β-actin. Data are presented as the mean ± SD from three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: NF-κB inhibition reduced SINV structural protein production in differentiated AP-7 cells. dAP-7 cells were infected with SINV and treated with either TPCA-1 (10 μM) or DMSO at 39°C. (A) Whole-cell lysates were probed by immunoblotting with antibodies against phosphorylated p65, IκBα, SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and β-actin. (B to D) Densitometric analysis of phosphorylated p65 (B), SINV capsid (C), and SINV nsP2 (D) normalized to β-actin. Data are presented as the mean ± SD from three independent experiments. *, P

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Inhibition, Infection

    SINV replication and induction of NF-κB activation in differentiated and cycling AP-7 cells. cAP-7 and dAP-7 cells were infected with SINV at 33°C and 39°C, respectively. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Reverse-phase protein array (RPPA) analysis of phosphorylated p65 (p-p65; S536) normalized to total p65 and IκBα normalized to β-actin in SINV-infected cAP-7 and dAP-7 cells. Values indicate the fold increase relative to the level in matched mock-infected samples. Data are presented as the mean ± SD for triplicate samples. **, P

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: SINV replication and induction of NF-κB activation in differentiated and cycling AP-7 cells. cAP-7 and dAP-7 cells were infected with SINV at 33°C and 39°C, respectively. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Reverse-phase protein array (RPPA) analysis of phosphorylated p65 (p-p65; S536) normalized to total p65 and IκBα normalized to β-actin in SINV-infected cAP-7 and dAP-7 cells. Values indicate the fold increase relative to the level in matched mock-infected samples. Data are presented as the mean ± SD for triplicate samples. **, P

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Activation Assay, Infection, Plaque Assay, Protein Array

    Effect of PKR inhibition results on SINV replication in mature neurons. dAP-7 cells were infected with SINV and treated with the PKR inhibitor C16 (1 μM) or DMSO at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Immunoblot analysis for phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), total eIF2α, and β-actin. (C) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: Effect of PKR inhibition results on SINV replication in mature neurons. dAP-7 cells were infected with SINV and treated with the PKR inhibitor C16 (1 μM) or DMSO at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Immunoblot analysis for phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), total eIF2α, and β-actin. (C) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Inhibition, Infection, Plaque Assay

    Effect of PKR absence on SINV replication in mature neurons. WT and PKR-deficient dAP-7 cells were infected with SINV at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Cell viability relative to day 0 (D0) cell counts, as determined by trypan blue exclusion. (C) Immunoblot analysis for PKR, phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and total eIF2α. (D) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

    Journal: Journal of Virology

    Article Title: NF-κB Activation Promotes Alphavirus Replication in Mature Neurons

    doi: 10.1128/JVI.01071-19

    Figure Lengend Snippet: Effect of PKR absence on SINV replication in mature neurons. WT and PKR-deficient dAP-7 cells were infected with SINV at 39°C. (A) Supernatants were assayed for infectious virus by plaque assay in BHK-21 cells. (B) Cell viability relative to day 0 (D0) cell counts, as determined by trypan blue exclusion. (C) Immunoblot analysis for PKR, phosphorylated eIF2α (S51), SINV nsP2, SINV structural proteins (pE2, E1/E2, capsid), and total eIF2α. (D) Densitometric analysis of phosphorylated eIF2α normalized to total eIF2α and SINV nsP2 and SINV pE2 normalized to β-actin. Data are presented as the mean ± SD and are representative of those from three independent experiments. *, P

    Article Snippet: Immunodetection was performed with the following antibodies diluted in TBS-T containing 5% bovine serum albumin (BSA; Sigma): monoclonal anti-phospho-p65 (antibody S536; 1:1,000; catalog number 3033), anti-p65 (1:1,000; catalog number 4764), anti-IκBα (1:1,000; catalog number 4814), anti-phospho-eIF2α (antibody S51; 1:1,000; catalog number 9721), anti-eIF2α (1:1,000; catalog number 9722), and anti-phospho-IKKα/β (antibody S176/177; 1:500; catalog number 2078) (all from Cell Signaling Technology); anti-PKR (1:500, catalog number sc-6282; Santa Cruz Biotechnology); polyclonal anti-SINV structural proteins (1:1,000) ( ); polyclonal anti-SINV nsP3 (1:1,000) ( ); and monoclonal anti-β-actin (1:10,000; catalog number MAB1501; EMD Millipore).

    Techniques: Infection, Plaque Assay

    Protein expression level of CAV2. (A) Western blotting revealed an increase in CAV2 level in Cx26 f/f P0Cre mice at postnatal day 21. (B) CAV level was normalized to that of β-actin and is expressed as relative to the amount present in each littermate control. Values represent the mean ± SE (n = 6 for control, n = 5 for Cx26 f/f P0Cre). **P = 2.5×10 −3 for CAV2, Student’s t -test.

    Journal: PLoS ONE

    Article Title: Deformation of the Outer Hair Cells and the Accumulation of Caveolin-2 in Connexin 26 Deficient Mice

    doi: 10.1371/journal.pone.0141258

    Figure Lengend Snippet: Protein expression level of CAV2. (A) Western blotting revealed an increase in CAV2 level in Cx26 f/f P0Cre mice at postnatal day 21. (B) CAV level was normalized to that of β-actin and is expressed as relative to the amount present in each littermate control. Values represent the mean ± SE (n = 6 for control, n = 5 for Cx26 f/f P0Cre). **P = 2.5×10 −3 for CAV2, Student’s t -test.

    Article Snippet: After blocking, each membrane was processed through sequential incubations with anti-CAV2 (1:500, Sigma Aldrich) and monoclonal anti-β-actin (1:1500; Sigma Aldrich) with horseradish peroxidase—conjugated anti-rabbit or anti-mouse IgG (1:40,000; GE Healthcare) as the secondary antibody.

    Techniques: Expressing, Western Blot, Mouse Assay