dusp4 mkp2 d9a5 rabbit mab  (Cell Signaling Technology Inc)

Journal: Autophagy

Article Title: The pro-oxidant adaptor p66SHC promotes B cell mitophagy by disrupting mitochondrial integrity and recruiting LC3-II

doi: 10.1080/15548627.2018.1505153

Figure Lengend Snippet: Commercial antibodies.

Article Snippet: Antibody Host Species Catalog number Source WB dilution IF dilution IP dilution Anti-ACTB mouse MAB1501 Merck Millipore 1:10,000 - - Anti-LC3B rabbit 3868 Cell Signaling Technology 1:500 1:100 1:500 Anti-MT-CO2 mouse Ab110258 Abcam 1:200 - - Anti-COX4I1/COXIV mouse Ab33985 Abcam 1:20,000 - - Anti-p-PRKAA/AMPKα rabbit 2535 Cell Signaling Technology 1:1000 1:100 - Anti- PRKAA/AMPKα rabbit 5832 Cell Signaling Technology 1:1000 - - Anti-p-MTOR rabbit 2448 Cell Signaling Technology 1:200 - - Anti-MTOR rabbit 2972 Cell Signaling Technology 1:250 - - Anti-Ubiquitin mouse 3936 Cell Signaling Technology 1:300 1:100 - Anti-PINK1 rabbit sc-33,796 Santa Cruz Biotechnology 1:500 - - Anti-GFP mouse A11120 Invitrogen 1:100 1:100 1:500 Anti-GFP rabbit A11122 Invitrogen 1:500 - 1:500 Anti-GOLGA2/GM130 mouse 610,822 BD Pharmingen 1:500 - - Anti-LAMP1 mouse 328,602 BioLegend 1:500 1:400 - Anti-PHB mouse CP34 Calbiochem 1:1000 - - Anti-PRKN rabbit sc-30,130 Santa Cruz Biotechnology - 1:10 - Anti-TOMM20 mouse sc-17,764 Santa Cruz Biotechnology 1:500 1:500 - Anti-TOMM20 rabbit sc-11,415 Santa Cruz Biotechnology 1:500 1:500 - Anti-BECN1 rabbit 3495 Cell Signaling Technology 1:500 - - Anti-PIK3C3/VPS34 rabbit D9A5 Cell Signaling Technology 1:1000 - - Anti-SHC rabbit 06–203 Merck Millipore 1:500 - 1:500 Anti-OPA1 mouse 612,606 BD Pharmingen 1:1000 - - Anti-MFN1 mouse ABC41 Merck Millipore 1:1000 - - Anti-DNM1L/DRP1 rabbit 611,112 BD Pharmingen 1:500 - - Anti-TIMM23 rabbit ab116329 Abcam 1:5000 - - Anti-HIF1A mouse 610,958 BD Pharmingen 1:2000 - - APC-anti-IgG1 mouse 560,089 BD Pharmingen - 1:50 - PE-anti-SDC1/CD138 mouse 142,503 BioLegend - 1:50 - Anti-RFP rabbit 600–401-379 Rockland Immunochemicals - 1:100 - Open in a separate window WB, western blot; IF, immunofluoscence; IP, immunoprecipitation Commercial antibodies.

Techniques:

Journal: eLife

Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

doi: 10.7554/elife.14023

Figure Lengend Snippet: Figure 1. Autophagy is activated on cytokine withdrawal in activated Tregs. (A) Immunoblots probed for LC3 in lysates of Tregs at onset (T0) and after 6 hr culture without IL-2. The values below are densitometry analysis of LC3II relative to tubulin. (B) Z-projected confocal images of Tregs at onset (T0) and cultured without IL-2 for times indicated and stained for LC3 (green) and Hoechst 33342 (blue). Change in fluorescence intensities for LC3 relative to T0 are plotted. (n=150 cells/time point). (C) Immunoblot probed for ATG5 in lysates of Tregs cultured as described in A. (DF) Apoptotic damage following 15 hr of IL-2 withdrawal in Tregs cultured in the presence of Bafilomycin (Baf) or 3-MA (D) or transduced with shRNA specific for VPS34 (E) or ATG7 (F) or a scrambled control (Scr). Immunoblots of scrambled and shRNA transfected cells are shown below. Data shown are the mean ± SD from at least 3 independent experiments, *p<0.03. Scale bar 5 mm. This figure is accompanied by Figure 1—figure supplement 1. DOI: 10.7554/eLife.14023.003 The following figure supplement is available for figure 1:

Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

Techniques: Western Blot, Cell Culture, Staining, Fluorescence, Transduction, shRNA, Control, Transfection

Journal: eLife

Article Title: Notch1 regulated autophagy controls survival and suppressor activity of activated murine T-regulatory cells

doi: 10.7554/elife.14023

Figure Lengend Snippet: Figure 5. Notch activity and autophagy regulate Treg suppressor function. (A) Representative, confocal images (central stack) of activated Tregs (upper panel), or T-effectors (lower panel) immune-stained for NIC (red) and Hoechst 33342 (blue). N: 15–20 cells/experiment. (B) Representative confocal (central stack) field views of activated Tregs immune-stained for Foxp3 (green) and Hoechst 33342 (blue). Tregs were derived from C57Bl/6 (wildtype, WT) or Cre negative (Cre-ve) or Cd4-Cre::Notch1lox/lox (Cre+ve) mice. (C) Real Time PCR quantification of genes enriched in Tregs, comparing activated Tregs from Cd4-Cre::Notch1lox/lox (open bars) and genetic control Cre-ive (black bars) mice. 5–6 mice are included in each group being compared. The data plotted is mean+/-SD. p**

Article Snippet: Primary antibodies used for western blot analysis include LC3 (D3U4C), Atg7 (D12B11), VPS34 (D9A5) Atg5 (D5F5U), Beclin-1 (D40C5) and Atg14 were from Cell Signaling Technology (used at a dilution of 1:500); NICD (clone mN1A) and DLL-1 (C-20) from Santa Cruz Biotechnology (used at adilution of 1:250); a-tubulin and actin (used at a dilution of 1:250) from Neomarker and Hes-1 (used at a dilution of 1:250) was from Millipore.

Techniques: Activity Assay, Staining, Derivative Assay, Real-time Polymerase Chain Reaction, Control, Expressing, Fluorescence, Generated, Isolation, Injection, Transduction, shRNA, Western Blot, Plasmid Preparation, Recombinant, In Vitro, Immunostaining, Flow Cytometry, Cell Culture

Journal: PLoS ONE

Article Title: Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

doi: 10.1371/journal.pone.0094634

Figure Lengend Snippet: ( A ) Co-IP analysis is used to study the interactions between RGS19 and RIP3, GNAI3 and RIP3, RGS19 and GNAI3. Myc-GNAI3 with Flag-RIP3 or Flag-RGS19, Flag-RIP3 with Myc-RGS19 were respectively co-expressed in 293T cells, Flag-RIP3 or Flag-RGS19 was then immunoprecipitated with anti-Flag M2-beads, and Myc-GNAI3 or Myc-RGS19 was detected in the immunoprecipitates with anti-myc antibody by Western blotting. ( B ) Knockdown of RGS19 or GNAI3 effectively blocked zVAD-induced cell death, but had no effect on TNF-induced cell death. Control and RGS19- or GNAI3-knockdown L929 cells were treated for 24 h with mock, TNF (10 ng/ml) or zVAD (20 µM). RIP1-, RIP3- or PI3KC3-knockdown L929 cells were also included. Then cell viabilities were measured (upper panel). The knockdown efficiencies were determined by relative mRNA levels calculated from real-time PCR results (lower panel). **, p<0.01.

Article Snippet: Rabbit anti-p-ERK, rabbit anti-ERK, rabbit anti-p38, rabbit anti-p-p38, rabbit anti-p-JNK and rabbit anti-PI3KC3 (D9A5) antibodies were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Knockdown, Control, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

doi: 10.1371/journal.pone.0094634

Figure Lengend Snippet: ( A–B ) Knockdown of RGS19 or GNAI3 inhibited zVAD-induced modification of LC3. Control and RGS19-knockdown ( A ) or GNAI3 knockdown ( B ) L929 cells were treated with mock or zVAD (20 µM) for 12 h. Cell lysates were subjected to western blot analysis with antibodies against LC3 and β-actin. Results from RIP1- or PI3KC3-knockdown cells were included as controls. ( C–D ) Knockdown of RGS19 or GNAI3 inhibited TNF-induced modification of LC3. Control and RGS19-knockdown ( C ) or GNAI3 knockdown ( D ) L929 cells were treated with mock or TNF (1 ng/ml) for 12 h. Cell lysates were subjected to western blot analysis with the indicated antibodies. ( E ) Knockdown of RGS19 or GNAI3 impaired zVAD-induced LC3 flux. Control and RGS19-knockdown or GNAI3-konckdown L929 cells were cultured with or without chloroquine (25 µM) and treated with or without zVAD (20 µM) for 12 h. LC3 levels were measured by Western blot.

Article Snippet: Rabbit anti-p-ERK, rabbit anti-ERK, rabbit anti-p38, rabbit anti-p-p38, rabbit anti-p-JNK and rabbit anti-PI3KC3 (D9A5) antibodies were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Knockdown, Modification, Control, Western Blot, Cell Culture

Journal: PLoS ONE

Article Title: Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

doi: 10.1371/journal.pone.0094634

Figure Lengend Snippet: ( A ) Knockdown of RGS19, GNAI3 or PI3KC3 had no effect on TNF-induced cell death in zVAD insensitive L929 subline. Control and RGS19- or GNAI3-knockdown L929 cells (left panel) and zVAD insensitive L929 cells (right panel) were treated for 24 h with mock, TNF (10 ng/ml) or zVAD (20 µM). Then cell viabilities were measured. ( B ) zVAD insensitive L929 subline had defect in RGS19-, GNAI3- and RIP3-dependent zVAD- or TNF-induced modification of LC3. Control and RGS19-, GNAI3- or RIP3-knockdown zVAD insensitive L929 cells were treated with mock, TNF (1 ng/ml) or zVAD (20 µM) for 12 h. Cell lysates were subjected to western blot analysis with antibodies against LC3 and β-actin. **, p<0.01.

Article Snippet: Rabbit anti-p-ERK, rabbit anti-ERK, rabbit anti-p38, rabbit anti-p-p38, rabbit anti-p-JNK and rabbit anti-PI3KC3 (D9A5) antibodies were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Knockdown, Control, Modification, Western Blot

Journal: PLoS ONE

Article Title: Regulator of G-Protein Signaling 19 (RGS19) and Its Partner Gα-Inhibiting Activity Polypeptide 3 (GNAI3) Are Required for zVAD-Induced Autophagy and Cell Death in L929 Cells

doi: 10.1371/journal.pone.0094634

Figure Lengend Snippet: ( A ) Knockdown of TNFR1 inhibited both TNF- and zVAD-induced cell deaths. Control and GFP- or TNFR1-knockdown L929 cells were treated with mock, TNF (10 ng/ml) or zVAD (20 µM) for 24 h. Then cell viabilities were measured. ( B ) Knockdown of TNF inhibited zVAD- but not TNF-induced cell death. Control and GFP- or TNF-knockdown L929 cells were treated with mock, TNF (10 ng/ml) or zVAD (20 µM) for 24 h. Then cell viabilities were measured. ( C ) Knockdown of RGS19, GNAI3, RIP3, or PI3KC3 all inhibited zVAD-induced increase of TNF mRNA. Control and RGS19-, GNAI3-, RIP3- or PI3KC3-knockdown L929 cells were treated with mock or zVAD (20 µM) for 3 h. TNF mRNA levels were measured by real-time PCR. ( D ) Knockdown of RGS19, GNAI3, RIP3, or PI3KC3 all inhibited zVAD-induced TNF secretion. Control and RGS19-, GNAI3-, RIP3- or PI3KC3-knockdown L929 cells were treated with mock or zVAD (20 µM) for 24 h. Then the cell culture medium was collected and concentrated 10 folds, and the TNF secretion was determined by ELISA. ( E ) Beclin-1, but not TNF or TNFR1, is required for zVAD-induced LC3 modification. Control and TNF-knockdown or TNFR-knockdown or Bclin 1-knockdown L929 cells were treated with mock or zVAD (20 µM) for 12 h. Cell lysates were subjected to Western blot analysis with antibodies against LC3 and β-actin. ( F ) Control and Bclin 1-knockdown L929 cells were treated with mock or zVAD (20 µM) for 12 h. Then TNF secretion was determined (left) and cell viabilities were measured (right). **, p<0.01.

Article Snippet: Rabbit anti-p-ERK, rabbit anti-ERK, rabbit anti-p38, rabbit anti-p-p38, rabbit anti-p-JNK and rabbit anti-PI3KC3 (D9A5) antibodies were obtained from Cell Signaling (Danvers, MA, USA).

Techniques: Knockdown, Control, Real-time Polymerase Chain Reaction, Cell Culture, Enzyme-linked Immunosorbent Assay, Modification, Western Blot