Structured Review

Millipore gapdh
Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and <t>ΔFosB.</t> (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls <t>(GAPDH)</t> are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p
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Images

1) Product Images from "Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB"

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2017.00924

Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p
Figure Legend Snippet: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p

Techniques Used: Western Blot, Injection

2) Product Images from "The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition"

Article Title: The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition

Journal: Pain

doi: 10.1016/j.pain.2009.11.017

Western blot analysis showing time course of CFA-induced expression of pJNK1, pJNK2, JNK1, and JNK2 in the spinal cord of rats. JNK1 and JNK2 are rapidly activated and maintained for > 14 days in both ipsilateral (A) and contralateral (B) lumbar spinal cord. Low panels in A and B show densities of pJNK1 and pJNK2 bands after being normalized to tubulin. Total levels of JNK1 and JNK2 do not change after inflammation (C). Low panels in C shows densities of JNK1 and JNK2 against GAPDH. *, P
Figure Legend Snippet: Western blot analysis showing time course of CFA-induced expression of pJNK1, pJNK2, JNK1, and JNK2 in the spinal cord of rats. JNK1 and JNK2 are rapidly activated and maintained for > 14 days in both ipsilateral (A) and contralateral (B) lumbar spinal cord. Low panels in A and B show densities of pJNK1 and pJNK2 bands after being normalized to tubulin. Total levels of JNK1 and JNK2 do not change after inflammation (C). Low panels in C shows densities of JNK1 and JNK2 against GAPDH. *, P

Techniques Used: Western Blot, Expressing

3) Product Images from "Dysfunctional transcripts are formed by alternative polyadenylation in OPMD"

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD

Journal: Oncotarget

doi: 10.18632/oncotarget.20640

Autophagy is hyper activated in OPMD muscle cell model Experiments were performed in stable muscle cell culture over-expressing wild type PABPN1 (A10) or expPABPN1 (A17). The expression of PABPN1 transgenes was induced by incubation with 2% HS. A. Western blot shows levels of transgenes A10 and A17 PABPN1-FLAG (55 kDa) and endogenous Pabpn1 (52 kDa). Autophagy activation is represented by LC3II and P62. Tubulin and Gapdh are used as loading controls. B. Images of representative immunofluorescence with anti-FLAG (green) and anti-LC3 (red) antibodies in cell cultures that were incubated with 2% HS for 3 hours or 48 hours. Scale bar is 10 µm. C. Chart bar shows mean LC3 puncta per nucleus in A10 or A17 culture. Mean and standard deviations are from 100 nuclei collected from three independent experiments. D. Bar chart shows mRNA levels of five ATG in A10 or A17 cultures. Fold change was obtained after normalisation to Hprt housekeeping gene and to parental culture. E. Bar chart shows the nuclear to cytosolic ratio of ATG transcripts from the distal primer set. F. Image shows a representative Western blot of nuclear and cytosolic fractions, marked by Emerin or Tubulin, respectively. G. Bar chart shows PABPN1 abundance in nuclear or cytosolic fractions in A10 or A17 cell cultures. PABPN1 abundance was calculated after normalization to loading control in each fraction. Averages and standard deviations are from 4 replicates. Averages and standard deviations are from three biological independent cultures. Statistical significance is assessed by the Student’s T-Test ( p
Figure Legend Snippet: Autophagy is hyper activated in OPMD muscle cell model Experiments were performed in stable muscle cell culture over-expressing wild type PABPN1 (A10) or expPABPN1 (A17). The expression of PABPN1 transgenes was induced by incubation with 2% HS. A. Western blot shows levels of transgenes A10 and A17 PABPN1-FLAG (55 kDa) and endogenous Pabpn1 (52 kDa). Autophagy activation is represented by LC3II and P62. Tubulin and Gapdh are used as loading controls. B. Images of representative immunofluorescence with anti-FLAG (green) and anti-LC3 (red) antibodies in cell cultures that were incubated with 2% HS for 3 hours or 48 hours. Scale bar is 10 µm. C. Chart bar shows mean LC3 puncta per nucleus in A10 or A17 culture. Mean and standard deviations are from 100 nuclei collected from three independent experiments. D. Bar chart shows mRNA levels of five ATG in A10 or A17 cultures. Fold change was obtained after normalisation to Hprt housekeeping gene and to parental culture. E. Bar chart shows the nuclear to cytosolic ratio of ATG transcripts from the distal primer set. F. Image shows a representative Western blot of nuclear and cytosolic fractions, marked by Emerin or Tubulin, respectively. G. Bar chart shows PABPN1 abundance in nuclear or cytosolic fractions in A10 or A17 cell cultures. PABPN1 abundance was calculated after normalization to loading control in each fraction. Averages and standard deviations are from 4 replicates. Averages and standard deviations are from three biological independent cultures. Statistical significance is assessed by the Student’s T-Test ( p

Techniques Used: Cell Culture, Expressing, Incubation, Western Blot, Activation Assay, Immunofluorescence

4) Product Images from "Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy"

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy

Journal: Nature Communications

doi: 10.1038/ncomms14454

Dystrophin expression in treated muscles improves muscle morphology. ( a ) TA muscles from treated mice were collected and analysed for expression of the mCherry reporter gene (top) or cryosectioned for immunostaining of dystrophin (bottom). Widespread dystrophin expression resulted from both strategies 1 and 2 (Scale bar, 1 mm). ( b ) Western analysis of muscles from treated and untreated mice (WT and mdx 4cv ) showing dystrophin (Dys), SpCas9, SaCas9 and GAPDH expression. Dystrophin was detected using antisera raised against the C terminus (CT); the SaCas9 nuclease carried an HA epitope tag to enable detection with anti-HA antibodies. ( c ) Quantification of GAPDH-normalized dystrophin expression in treated TAs compared with WT muscles ( n =4). ( d ) Immunostained cross-sections from treated and control mice were analysed for the percentage of all myofibers expressing dystrophin ( n =5). ( e ) Shown is the cross-sectional area (CXA) size distribution of individual myofibers from treated and control muscles ( n > 12,500 total fibres per group). ( f ) The total myogenic cross-sectional area (CXA) that was dystrophin-positive is shown for treated and WT control muscles ( n =5). ( g ) Individual myofiber size distribution for treated TAs relative to dystrophin expression. ( h ) The percentage of myofibers containing centrally located nuclei is shown for dystrophin-positive treated myofibers and for total myofibers of control TA muscles ( n =5). Data are shown as mean±s.e.m. *** P
Figure Legend Snippet: Dystrophin expression in treated muscles improves muscle morphology. ( a ) TA muscles from treated mice were collected and analysed for expression of the mCherry reporter gene (top) or cryosectioned for immunostaining of dystrophin (bottom). Widespread dystrophin expression resulted from both strategies 1 and 2 (Scale bar, 1 mm). ( b ) Western analysis of muscles from treated and untreated mice (WT and mdx 4cv ) showing dystrophin (Dys), SpCas9, SaCas9 and GAPDH expression. Dystrophin was detected using antisera raised against the C terminus (CT); the SaCas9 nuclease carried an HA epitope tag to enable detection with anti-HA antibodies. ( c ) Quantification of GAPDH-normalized dystrophin expression in treated TAs compared with WT muscles ( n =4). ( d ) Immunostained cross-sections from treated and control mice were analysed for the percentage of all myofibers expressing dystrophin ( n =5). ( e ) Shown is the cross-sectional area (CXA) size distribution of individual myofibers from treated and control muscles ( n > 12,500 total fibres per group). ( f ) The total myogenic cross-sectional area (CXA) that was dystrophin-positive is shown for treated and WT control muscles ( n =5). ( g ) Individual myofiber size distribution for treated TAs relative to dystrophin expression. ( h ) The percentage of myofibers containing centrally located nuclei is shown for dystrophin-positive treated myofibers and for total myofibers of control TA muscles ( n =5). Data are shown as mean±s.e.m. *** P

Techniques Used: Expressing, Mouse Assay, Immunostaining, Western Blot

5) Product Images from "BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells"

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2018.01.001

Induction of prosurvival autophagy in chemoresistant cells and pharmacological inhibition of autophagy augments the sensitivity of chemoresistant breast cancer cells (A) Basal level of autophagy was increased in chemoresistant cells as LC3-II, ATG5 and Beclin-1 proteins expression were increased in chemoresistant cells compared to their respective parental cells in western blot analysis. (B) Autophagic flux was determined by the accumulation of LC3-II and p62 after combined treatment of DOX (10 ng/ml) for 72 h and autophagic flux inhibitor Baf A1 (25 nM) for 4 h in both the BT-549Par and BT-549 r DOX 20 cells and (C) similarly in MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines after combined treatment of 5-FU (1 μg/ml) for 72 h and Baf A1 (25 nM) for 4 h. GAPDH was used as loading control. (D) Breast cancer cell lines BT-549Par and BT-549 r DOX 20 were treated with 10 ng/ml and 80 ng/ml of DOX for 72 h with or without Baf A1 (25 nM for 4 h), (E) similarly MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines were treated with 1 μg/ml and 18 μg/ml of 5-FU for 72 h with or without Baf A1 (25 nM for 4 h). Total cell death was quantified by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p
Figure Legend Snippet: Induction of prosurvival autophagy in chemoresistant cells and pharmacological inhibition of autophagy augments the sensitivity of chemoresistant breast cancer cells (A) Basal level of autophagy was increased in chemoresistant cells as LC3-II, ATG5 and Beclin-1 proteins expression were increased in chemoresistant cells compared to their respective parental cells in western blot analysis. (B) Autophagic flux was determined by the accumulation of LC3-II and p62 after combined treatment of DOX (10 ng/ml) for 72 h and autophagic flux inhibitor Baf A1 (25 nM) for 4 h in both the BT-549Par and BT-549 r DOX 20 cells and (C) similarly in MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines after combined treatment of 5-FU (1 μg/ml) for 72 h and Baf A1 (25 nM) for 4 h. GAPDH was used as loading control. (D) Breast cancer cell lines BT-549Par and BT-549 r DOX 20 were treated with 10 ng/ml and 80 ng/ml of DOX for 72 h with or without Baf A1 (25 nM for 4 h), (E) similarly MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines were treated with 1 μg/ml and 18 μg/ml of 5-FU for 72 h with or without Baf A1 (25 nM for 4 h). Total cell death was quantified by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p

Techniques Used: Inhibition, Expressing, Western Blot, Multiple Displacement Amplification, Double Staining, Flow Cytometry, Cytometry

6) Product Images from "Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines"

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines

Journal: PLoS ONE

doi: 10.1371/journal.pone.0081747

Expression profile of MSK1 and p-MSK1 (Thr-581 and Ser-360) following LPS intracerebral injection. A . Protein levels of t-MSK1, p-MSK1 Thr-581, p-MSK1 Ser-360 were detected before (control) and after injury. GAPDH was also detected by Western blotting. B . Quantification graphs (relative optical density) of the intensity of staining of p-MSK1 (Thr-581) and total MSK1 to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. C–H . Immunofluorescence staining of MSK1 and p-MSk1 (Thr581) was performed to assess the staining changes for MSK1 and p-MSK1 immunoreactivity in the cortex at day 1 after LPS-injection. I . Negative control. * and # indicate significant differences at P
Figure Legend Snippet: Expression profile of MSK1 and p-MSK1 (Thr-581 and Ser-360) following LPS intracerebral injection. A . Protein levels of t-MSK1, p-MSK1 Thr-581, p-MSK1 Ser-360 were detected before (control) and after injury. GAPDH was also detected by Western blotting. B . Quantification graphs (relative optical density) of the intensity of staining of p-MSK1 (Thr-581) and total MSK1 to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. C–H . Immunofluorescence staining of MSK1 and p-MSk1 (Thr581) was performed to assess the staining changes for MSK1 and p-MSK1 immunoreactivity in the cortex at day 1 after LPS-injection. I . Negative control. * and # indicate significant differences at P

Techniques Used: Expressing, Injection, Western Blot, Staining, Immunofluorescence, Negative Control

Expression of pro-inflammatory mediators by active astrocytes after LPS treatment. A . Western blot analysis showed the time course of TNFα, iNOS, and GFAP protein expression in LPS-treated rats. B . Quantification graphs (relative optical density) of the intensity of staining of iNOS, TNFα, and GFAP to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. The data are means ± SEM ( n = 3, **, P
Figure Legend Snippet: Expression of pro-inflammatory mediators by active astrocytes after LPS treatment. A . Western blot analysis showed the time course of TNFα, iNOS, and GFAP protein expression in LPS-treated rats. B . Quantification graphs (relative optical density) of the intensity of staining of iNOS, TNFα, and GFAP to GAPDH at each time point. GAPDH was used to confirm that equal amounts of protein were run on the gel. The data are means ± SEM ( n = 3, **, P

Techniques Used: Expressing, Western Blot, Staining

7) Product Images from "Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB"

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2017.00924

Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p
Figure Legend Snippet: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p

Techniques Used: Western Blot, Injection

8) Product Images from "BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells"

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2018.01.001

Induction of prosurvival autophagy in chemoresistant cells and pharmacological inhibition of autophagy augments the sensitivity of chemoresistant breast cancer cells (A) Basal level of autophagy was increased in chemoresistant cells as LC3-II, ATG5 and Beclin-1 proteins expression were increased in chemoresistant cells compared to their respective parental cells in western blot analysis. (B) Autophagic flux was determined by the accumulation of LC3-II and p62 after combined treatment of DOX (10 ng/ml) for 72 h and autophagic flux inhibitor Baf A1 (25 nM) for 4 h in both the BT-549Par and BT-549 r DOX 20 cells and (C) similarly in MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines after combined treatment of 5-FU (1 μg/ml) for 72 h and Baf A1 (25 nM) for 4 h. GAPDH was used as loading control. (D) Breast cancer cell lines BT-549Par and BT-549 r DOX 20 were treated with 10 ng/ml and 80 ng/ml of DOX for 72 h with or without Baf A1 (25 nM for 4 h), (E) similarly MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines were treated with 1 μg/ml and 18 μg/ml of 5-FU for 72 h with or without Baf A1 (25 nM for 4 h). Total cell death was quantified by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p
Figure Legend Snippet: Induction of prosurvival autophagy in chemoresistant cells and pharmacological inhibition of autophagy augments the sensitivity of chemoresistant breast cancer cells (A) Basal level of autophagy was increased in chemoresistant cells as LC3-II, ATG5 and Beclin-1 proteins expression were increased in chemoresistant cells compared to their respective parental cells in western blot analysis. (B) Autophagic flux was determined by the accumulation of LC3-II and p62 after combined treatment of DOX (10 ng/ml) for 72 h and autophagic flux inhibitor Baf A1 (25 nM) for 4 h in both the BT-549Par and BT-549 r DOX 20 cells and (C) similarly in MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines after combined treatment of 5-FU (1 μg/ml) for 72 h and Baf A1 (25 nM) for 4 h. GAPDH was used as loading control. (D) Breast cancer cell lines BT-549Par and BT-549 r DOX 20 were treated with 10 ng/ml and 80 ng/ml of DOX for 72 h with or without Baf A1 (25 nM for 4 h), (E) similarly MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cell lines were treated with 1 μg/ml and 18 μg/ml of 5-FU for 72 h with or without Baf A1 (25 nM for 4 h). Total cell death was quantified by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p

Techniques Used: Inhibition, Expressing, Western Blot, Multiple Displacement Amplification, Double Staining, Flow Cytometry, Cytometry

9) Product Images from "In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport"

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

Journal: Infection and Immunity

doi: 10.1128/IAI.00322-15

Impact of Waddlia chondrophila infection on integrity of host cell proteins. (A) HeLa cells were infected with W. chondrophila at an MOI of 1 and whole-cell lysates were prepared with either RIPA (left) or urea (right) extraction buffer (see Materials and Methods) at 30 and 48 h p.i. Ten micrograms of protein was loaded onto each lane. Shown are representative Western blots of uninfected or W. chondrophila -infected cells. The depicted results were reproduced independently. RFX5, n = 3 (urea) and 5 (RIPA); cyclin B1, n = 2 (urea) and 4 (RIPA); Bim EL , n = 4 (urea) and 6 (RIPA); USF-1, n = 3 (urea) and 3 (RIPA); p65, n = 3 (urea) and 4 (RIPA); vimentin, n = 3 (urea) and 6 (RIPA); cytokeratin 8 (CK 8), n = 3 (urea) and 5 (RIPA). Actin, tubulin, and GAPDH were used as loading controls. (B) Representative Western blots of uninfected, W. chondrophila -infected, and C. trachomatis -infected cells. Urea lysates were prepared at 24, 30, or 40 h p.i.
Figure Legend Snippet: Impact of Waddlia chondrophila infection on integrity of host cell proteins. (A) HeLa cells were infected with W. chondrophila at an MOI of 1 and whole-cell lysates were prepared with either RIPA (left) or urea (right) extraction buffer (see Materials and Methods) at 30 and 48 h p.i. Ten micrograms of protein was loaded onto each lane. Shown are representative Western blots of uninfected or W. chondrophila -infected cells. The depicted results were reproduced independently. RFX5, n = 3 (urea) and 5 (RIPA); cyclin B1, n = 2 (urea) and 4 (RIPA); Bim EL , n = 4 (urea) and 6 (RIPA); USF-1, n = 3 (urea) and 3 (RIPA); p65, n = 3 (urea) and 4 (RIPA); vimentin, n = 3 (urea) and 6 (RIPA); cytokeratin 8 (CK 8), n = 3 (urea) and 5 (RIPA). Actin, tubulin, and GAPDH were used as loading controls. (B) Representative Western blots of uninfected, W. chondrophila -infected, and C. trachomatis -infected cells. Urea lysates were prepared at 24, 30, or 40 h p.i.

Techniques Used: Infection, Western Blot

10) Product Images from "Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice"

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice

Journal: Scientific Reports

doi: 10.1038/srep46019

Expression analysis of mice with mutations in eEF1A2. ( A ) Representative western blot showing eEF1A2 expression in protein extracts from brains of mutant founder mice from the CRISPR gene targeting experiment. The negative control was a brain extract from a wasted mouse (denoted −/−) and the loading control was GAPDH. The blot images are cropped for clarity and a larger area is shown in the Supplementary Data File . ( B ) Quantification of eEF1A2 expression in brain samples from mice with key genotypes. Controls were aged matched wild-type mice that were offspring of the F0+/− heterozygous mice. Expression levels are derived from three repeats of the Western analysis, normalised to GAPDH and expressed relative to the average obtained from three independent wild-type controls. **Indicates p
Figure Legend Snippet: Expression analysis of mice with mutations in eEF1A2. ( A ) Representative western blot showing eEF1A2 expression in protein extracts from brains of mutant founder mice from the CRISPR gene targeting experiment. The negative control was a brain extract from a wasted mouse (denoted −/−) and the loading control was GAPDH. The blot images are cropped for clarity and a larger area is shown in the Supplementary Data File . ( B ) Quantification of eEF1A2 expression in brain samples from mice with key genotypes. Controls were aged matched wild-type mice that were offspring of the F0+/− heterozygous mice. Expression levels are derived from three repeats of the Western analysis, normalised to GAPDH and expressed relative to the average obtained from three independent wild-type controls. **Indicates p

Techniques Used: Expressing, Mouse Assay, Western Blot, Mutagenesis, CRISPR, Negative Control, Derivative Assay

Related Articles

Centrifugation:

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: The samples were then homogenized in lysis buffer (1% NP-40, 50 mmol/L Tris, pH 7.5, 5 mmol/L EDTA, 1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mmol/L PMSF, 10 µg/mL aprotinin, and 1 µg/mL leupeptin) and clarified by centrifugation (20 min in a microcentrifuge, 4°C). .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

Expressing:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA). .. Expression levels of the target protein bands were normalized to β-actin, GAPDH or LAMP1 (lysosomal fraction), and expressed as a fold of sham control.

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT. .. Differences in expression between mice with the G70S/−, − / − and +/+ genotypes were assessed by one way ANOVA with tukey post-hoc testing.

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: Antibodies and genetic constructs Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. .. VSV-G and pFIV-34N were kind gifts from Todd Waldman, Georgetown University. siRNA and shRNA expression constructs were obtained from Open Biosystems.

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. .. VSV-G and pFIV-34N were kind gifts from Todd Waldman, Georgetown University. siRNA and shRNA expression constructs were obtained from Open Biosystems.

Polymerase Chain Reaction:

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: .. Antibodies and genetic constructs Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. ..

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: .. Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. ..

Bradford Assay:

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: After determination of the protein concentration using the Bradford assay (Bio-Rad), the resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

Construct:

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: .. Antibodies and genetic constructs Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. ..

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: Paragraph title: Antibodies and genetic constructs ... Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used.

Incubation:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Membranes were incubated with primary antibodies overnight, with HRP-conjugated secondary antibodies for 1 h, visualized using SuperSignal West Dura Extended Duration Substrate (ThermoFisher), and imaged with ChemIDoc TM MP system (Bio-Rad). .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain
Article Snippet: .. The blots were blocked and incubated overnight at 4 °C with antibodies against CASP6 (rabbit, 1:1000, Cell Signaling) and GAPDH (mouse, 1:20000, Millipore). .. These blots were incubated further with HRP-conjugated secondary antibody and developed in ECL solution.

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot. .. Blots were the incubated in secondary antibodies conjugated with IRDye 800CW or IRDye 680RD (Licor, NE, USA).

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells
Article Snippet: .. After blocking in 5% BSA for 1 h at room temperature, the nitrocellulose membranes were incubated overnight at 4°C with primary antibodies directed against BAG3 (rabbit, 1:5000, Abnova, Heidelberg, Germany), LC3 (rabbit, 1:1000, Thermo Fisher Scientific, Rockford, USA), p62 (mouse, 1:1000, BD Biosciences, USA), GAPDH (mouse, 1:10,000, Calbiochem) and rest were against Bcl-2, Mcl-1, Bcl-xL, HSP70, HSF-1, Bak, Bax, ATG5, Beclin-1, pFAK (Tyr397), FAK, HSP60, E-cadherin and N-cadherin which were purchased from Cell Signaling Technology (Danvers, USA) raised in rabbit and used in the dilution of 1:1000. .. Following incubation, primary antibodies were detected by using respective secondary antibodies coupled with infrared dyes in green (800 CW) or red (680 RD) (IRDye goat anti-rabbit or anti-mouse from LICOR Biosciences, Bad Homburg, Germany diluted in 1:10,000 in 3% BSA for 1 h at room temperature and the signals were detected using the LI-COR Odyssey Infrared Imager (LI-COR Biosciences, Bad Homburg, Germany).

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000). .. Horseradish-peroxidase conjugated secondary antibody staining (1:50,000) was performed for 1 h at room temperature before signal development using Clarity Western ECL substrate (BioRad) and visualization using a Chemidoc MP imaging system (BioRad).

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: .. Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT. .. Images were analysed using ImageJ software to assess band intensity and values expressed relative to the loading control.

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport
Article Snippet: After blocking of the membranes (5% milk in Tris-buffered saline–Tween [TBS-T]), they were incubated with the respective antibody at 4°C overnight. .. Primary antibodies were directed against β-actin (mouse, 1:10,000; Sigma no. A5441), Bim C34C5 (rabbit, 1:5,000; Cell Signaling no. 2933), vimentin (rabbit, 1:1,000; Acris no. AP00289PU-N), NF-κB p65 (rabbit, 1:5,000; Cell Signaling no. 4764), cytokeratin-8 (1:20,.000; Acris no. BM5045P), RFX5 (rabbit, 1:1,000; Rockland no. 200-401-194), USF-1 (rabbit, 1:2,000; Santa Cruz no. sc-8983), Cyclin B1 (mouse, 1:2,000; Cell Signaling no. 4135), GAPDH (mouse, 1:10,000; Millipore no. MAB374) and α-tubulin (mouse, 1:10,000; Sigma no. T9026).

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma). .. After incubating with an anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody, proteins were visualized using an enhanced chemiluminescence system (ECL, Pierce Company, USA).

Article Title: The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition
Article Snippet: .. The blots were blocked with 5% milk and incubated overnight at 4°C with antibody against phosphorylated JNK (pJNK, rabbit, 1:1000; Cell Signaling), phosphorylated c-Jun (rabbit, 1:300; Cell Signaling), JNK (rabbit, 1:1000; Cell Signaling), GAPDH (mouse, 1:20000; Millipore), and β-tubulin (mouse, 1:5000; Sigma). .. These blots were further incubated with HRP-conjugated secondary antibody, developed in ECL solution, and exposed onto Hyperfilm (Amersham Biosciences) for 1–10 min.

Mass Spectrometry:

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: .. The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot. .. Total RNA in fractions was assessed using Bioanalyzer RNA kits (Agilent Genomics).

BIA-KA:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Protein concentration was determined by the Pierce BCA method (ThermoFisher Scientific, US). .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Muscle proteins were extracted in radioimmunoprecipitation analysis buffer (RIPA) supplemented with 5 mM EDTA and 3% protease inhibitor cocktail (Sigma, Cat# P8340), for 1 hour on ice with gentle agitation every 15 min. Total protein concentration was determined using Pierce BCA assay kit (Thermo Fisher). .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Western Blot:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Paragraph title: Western blot analysis ... Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain
Article Snippet: Paragraph title: Western Blot ... The blots were blocked and incubated overnight at 4 °C with antibodies against CASP6 (rabbit, 1:1000, Cell Signaling) and GAPDH (mouse, 1:20000, Millipore).

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: .. The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot. .. Total RNA in fractions was assessed using Bioanalyzer RNA kits (Agilent Genomics).

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells
Article Snippet: Paragraph title: SDS-PAGE and Western Blotting ... After blocking in 5% BSA for 1 h at room temperature, the nitrocellulose membranes were incubated overnight at 4°C with primary antibodies directed against BAG3 (rabbit, 1:5000, Abnova, Heidelberg, Germany), LC3 (rabbit, 1:1000, Thermo Fisher Scientific, Rockford, USA), p62 (mouse, 1:1000, BD Biosciences, USA), GAPDH (mouse, 1:10,000, Calbiochem) and rest were against Bcl-2, Mcl-1, Bcl-xL, HSP70, HSF-1, Bak, Bax, ATG5, Beclin-1, pFAK (Tyr397), FAK, HSP60, E-cadherin and N-cadherin which were purchased from Cell Signaling Technology (Danvers, USA) raised in rabbit and used in the dilution of 1:1000.

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000). .. Horseradish-peroxidase conjugated secondary antibody staining (1:50,000) was performed for 1 h at room temperature before signal development using Clarity Western ECL substrate (BioRad) and visualization using a Chemidoc MP imaging system (BioRad).

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport
Article Snippet: Paragraph title: Western blotting. ... Primary antibodies were directed against β-actin (mouse, 1:10,000; Sigma no. A5441), Bim C34C5 (rabbit, 1:5,000; Cell Signaling no. 2933), vimentin (rabbit, 1:1,000; Acris no. AP00289PU-N), NF-κB p65 (rabbit, 1:5,000; Cell Signaling no. 4764), cytokeratin-8 (1:20,.000; Acris no. BM5045P), RFX5 (rabbit, 1:1,000; Rockland no. 200-401-194), USF-1 (rabbit, 1:2,000; Santa Cruz no. sc-8983), Cyclin B1 (mouse, 1:2,000; Cell Signaling no. 4135), GAPDH (mouse, 1:10,000; Millipore no. MAB374) and α-tubulin (mouse, 1:10,000; Sigma no. T9026).

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: Paragraph title: Western Blotting Analysis ... The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

Article Title: The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition
Article Snippet: Paragraph title: 2.3 Western blots ... The blots were blocked with 5% milk and incubated overnight at 4°C with antibody against phosphorylated JNK (pJNK, rabbit, 1:1000; Cell Signaling), phosphorylated c-Jun (rabbit, 1:300; Cell Signaling), JNK (rabbit, 1:1000; Cell Signaling), GAPDH (mouse, 1:20000; Millipore), and β-tubulin (mouse, 1:5000; Sigma).

Immunohistochemistry:

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
Article Snippet: .. The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
Article Snippet: .. Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. Proximity ligation assay (PLA) To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

Protease Inhibitor:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Western blot analysis Mouse spinal cord tissue (5 mm) centered on the injury site was lysed and homogenized in RIPA buffer (Sigma-Aldrich) supplemented with 1 × protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail II and III (Sigma-Aldrich), sonicated and centrifuged at 20,000 × g for 20 min , . .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: The protease inhibitor cocktail (SigmaFAST protease inhibitor; Sigma-Aldrich) was freshly added. .. The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot.

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Muscle proteins were extracted in radioimmunoprecipitation analysis buffer (RIPA) supplemented with 5 mM EDTA and 3% protease inhibitor cocktail (Sigma, Cat# P8340), for 1 hour on ice with gentle agitation every 15 min. Total protein concentration was determined using Pierce BCA assay kit (Thermo Fisher). .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Infection:

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport
Article Snippet: For Western blot analysis, 200,000 HeLa cells were seeded in 6-well plates and infected as described above. .. Primary antibodies were directed against β-actin (mouse, 1:10,000; Sigma no. A5441), Bim C34C5 (rabbit, 1:5,000; Cell Signaling no. 2933), vimentin (rabbit, 1:1,000; Acris no. AP00289PU-N), NF-κB p65 (rabbit, 1:5,000; Cell Signaling no. 4764), cytokeratin-8 (1:20,.000; Acris no. BM5045P), RFX5 (rabbit, 1:1,000; Rockland no. 200-401-194), USF-1 (rabbit, 1:2,000; Santa Cruz no. sc-8983), Cyclin B1 (mouse, 1:2,000; Cell Signaling no. 4135), GAPDH (mouse, 1:10,000; Millipore no. MAB374) and α-tubulin (mouse, 1:10,000; Sigma no. T9026).

Imaging:

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000). .. Horseradish-peroxidase conjugated secondary antibody staining (1:50,000) was performed for 1 h at room temperature before signal development using Clarity Western ECL substrate (BioRad) and visualization using a Chemidoc MP imaging system (BioRad).

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: Total protein was then imaged on the Odyssey Fc Imaging System (LI-COR) and membranes were blocked for 1 hour in Odyssey blocking buffer (LI-COR). .. Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT.

Protein Concentration:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Protein concentration was determined by the Pierce BCA method (ThermoFisher Scientific, US). .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Muscle proteins were extracted in radioimmunoprecipitation analysis buffer (RIPA) supplemented with 5 mM EDTA and 3% protease inhibitor cocktail (Sigma, Cat# P8340), for 1 hour on ice with gentle agitation every 15 min. Total protein concentration was determined using Pierce BCA assay kit (Thermo Fisher). .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: After determination of the protein concentration using the Bradford assay (Bio-Rad), the resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

Reverse Transcription Polymerase Chain Reaction:

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Specific indel mutations or deletions in the dystrophin transcript were identified by Sanger sequencing of individual subclones of RT–PCR fragments. .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Sonication:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Western blot analysis Mouse spinal cord tissue (5 mm) centered on the injury site was lysed and homogenized in RIPA buffer (Sigma-Aldrich) supplemented with 1 × protease inhibitor cocktail (Sigma-Aldrich) and phosphatase inhibitor cocktail II and III (Sigma-Aldrich), sonicated and centrifuged at 20,000 × g for 20 min , . .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: Samples were sonicated for 3x10 second pulses at 1MHz. .. The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot.

Nucleic Acid Electrophoresis:

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Muscle lysates from WT (10 and 1 μg), untreated mdx 4cv (30 μg) and treated mdx 4cv (30 μg) mice were denatured at 99 degrees Celsius for 10 min, quenched on ice and separated via gel electrophoresis after loading onto Bolt 4–12% Bis-Tris polyacrylamide gels (Invitrogen). .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: After determination of the protein concentration using the Bradford assay (Bio-Rad), the resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

Isolation:

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: Following subcellular fractionation, RNA was isolated from each fraction as described above. .. The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot.

Mouse Assay:

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Muscle lysates from WT (10 and 1 μg), untreated mdx 4cv (30 μg) and treated mdx 4cv (30 μg) mice were denatured at 99 degrees Celsius for 10 min, quenched on ice and separated via gel electrophoresis after loading onto Bolt 4–12% Bis-Tris polyacrylamide gels (Invitrogen). .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT. .. Differences in expression between mice with the G70S/−, − / − and +/+ genotypes were assessed by one way ANOVA with tukey post-hoc testing.

Sequencing:

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Specific indel mutations or deletions in the dystrophin transcript were identified by Sanger sequencing of individual subclones of RT–PCR fragments. .. Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000).

Blocking Assay:

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells
Article Snippet: .. After blocking in 5% BSA for 1 h at room temperature, the nitrocellulose membranes were incubated overnight at 4°C with primary antibodies directed against BAG3 (rabbit, 1:5000, Abnova, Heidelberg, Germany), LC3 (rabbit, 1:1000, Thermo Fisher Scientific, Rockford, USA), p62 (mouse, 1:1000, BD Biosciences, USA), GAPDH (mouse, 1:10,000, Calbiochem) and rest were against Bcl-2, Mcl-1, Bcl-xL, HSP70, HSF-1, Bak, Bax, ATG5, Beclin-1, pFAK (Tyr397), FAK, HSP60, E-cadherin and N-cadherin which were purchased from Cell Signaling Technology (Danvers, USA) raised in rabbit and used in the dilution of 1:1000. .. Following incubation, primary antibodies were detected by using respective secondary antibodies coupled with infrared dyes in green (800 CW) or red (680 RD) (IRDye goat anti-rabbit or anti-mouse from LICOR Biosciences, Bad Homburg, Germany diluted in 1:10,000 in 3% BSA for 1 h at room temperature and the signals were detected using the LI-COR Odyssey Infrared Imager (LI-COR Biosciences, Bad Homburg, Germany).

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: .. Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT. .. Images were analysed using ImageJ software to assess band intensity and values expressed relative to the loading control.

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport
Article Snippet: After blocking of the membranes (5% milk in Tris-buffered saline–Tween [TBS-T]), they were incubated with the respective antibody at 4°C overnight. .. Primary antibodies were directed against β-actin (mouse, 1:10,000; Sigma no. A5441), Bim C34C5 (rabbit, 1:5,000; Cell Signaling no. 2933), vimentin (rabbit, 1:1,000; Acris no. AP00289PU-N), NF-κB p65 (rabbit, 1:5,000; Cell Signaling no. 4764), cytokeratin-8 (1:20,.000; Acris no. BM5045P), RFX5 (rabbit, 1:1,000; Rockland no. 200-401-194), USF-1 (rabbit, 1:2,000; Santa Cruz no. sc-8983), Cyclin B1 (mouse, 1:2,000; Cell Signaling no. 4135), GAPDH (mouse, 1:10,000; Millipore no. MAB374) and α-tubulin (mouse, 1:10,000; Sigma no. T9026).

Polyacrylamide Gel Electrophoresis:

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: After determination of the protein concentration using the Bradford assay (Bio-Rad), the resulting supernatant was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

Lysis:

Article Title: Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain
Article Snippet: Western Blot Protein samples were prepared from dorsal root ganglia by homogenization in a lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich), as previously described . .. The blots were blocked and incubated overnight at 4 °C with antibodies against CASP6 (rabbit, 1:1000, Cell Signaling) and GAPDH (mouse, 1:20000, Millipore).

Article Title: Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines
Article Snippet: After appropriate stimulation, cells were washed twice with ice-cold D-Hanks and extracted in lysis buffer for 45 min on ice. .. The membrane was then blocked with 5% nonfat milk and incubated with primary antibodies against MSK1 (rabbit, 1∶1000; Novus Biologicals), Phospho-MSK1 (Thr581) (rabbit, 1∶1000; Cell-Signalling), Phospho-MSK1 (Ser360) (rabbit, 1∶5000; abcam), iNOS, TNFα, GFAP (mouse, 1∶1000; Sigma), or GAPDH (mouse, 1∶1000; Sigma).

IA:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA). .. Expression levels of the target protein bands were normalized to β-actin, GAPDH or LAMP1 (lysosomal fraction), and expressed as a fold of sham control.

SDS Page:

Article Title: Lysosomal damage after spinal cord injury causes accumulation of RIPK1 and RIPK3 proteins and potentiation of necroptosis
Article Snippet: Samples were run on 4–20% SDS–PAGE (Bio-Rad, Hercules, CA), and transferred to PVDF membrane (Bio-Rad). .. Primary antibodies: LC3 (1:1000, Novus, Cat. No. NB100-2220), p62/SQSTM1 (1:1000; BD Bioscience, Cat. No. 610832), CTSD (1:100, Santa Cruz Biotechnology, Cat. No. sc-6486), RIPK1 (1:1000; Cell Signaling, Cat. No. 3493, Danvers, MA), RIPK3 (1:1000, ProSci, Cat. No. 2283, Poway, CA), MLKL (1:1000, EMD Millipore, Cat. No. MABC604, Temecula, CA), β-Actin (1:10 000; Sigma-Aldrich, Cat. No. A1978), Fordin/SPTAN1 (1:5000; Enzo Life Science International, Cat. No. BML-FG6090), GAPDH (1:2000; Millipore, Cat. No. AB2302), LAMP1 (1:1000; Cat. No. 1D4B, developed by J. Thomas August, Developmental Studies Hybridoma Bank/NICHD, maintained by The University of Iowa, Department of Biology, Iowa City, IA).

Article Title: Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain
Article Snippet: Samples were separated on SDS–PAGE gel and transferred to nitrocellulose blots. .. The blots were blocked and incubated overnight at 4 °C with antibodies against CASP6 (rabbit, 1:1000, Cell Signaling) and GAPDH (mouse, 1:20000, Millipore).

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells
Article Snippet: Paragraph title: SDS-PAGE and Western Blotting ... After blocking in 5% BSA for 1 h at room temperature, the nitrocellulose membranes were incubated overnight at 4°C with primary antibodies directed against BAG3 (rabbit, 1:5000, Abnova, Heidelberg, Germany), LC3 (rabbit, 1:1000, Thermo Fisher Scientific, Rockford, USA), p62 (mouse, 1:1000, BD Biosciences, USA), GAPDH (mouse, 1:10,000, Calbiochem) and rest were against Bcl-2, Mcl-1, Bcl-xL, HSP70, HSF-1, Bak, Bax, ATG5, Beclin-1, pFAK (Tyr397), FAK, HSP60, E-cadherin and N-cadherin which were purchased from Cell Signaling Technology (Danvers, USA) raised in rabbit and used in the dilution of 1:1000.

Article Title: The c-Jun N-terminal Kinase 1 (JNK1) in spinal astrocytes is required for the maintenance of bilateral mechanical allodynia under a persistent inflammatory pain condition
Article Snippet: Protein samples (25 μg) were separated on SDS-PAGE gel and transferred to nitrocellulose blots. .. The blots were blocked with 5% milk and incubated overnight at 4°C with antibody against phosphorylated JNK (pJNK, rabbit, 1:1000; Cell Signaling), phosphorylated c-Jun (rabbit, 1:300; Cell Signaling), JNK (rabbit, 1:1000; Cell Signaling), GAPDH (mouse, 1:20000; Millipore), and β-tubulin (mouse, 1:5000; Sigma).

Plasmid Preparation:

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: .. Antibodies and genetic constructs Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. ..

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: .. Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. ..

Software:

Article Title: Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain
Article Snippet: The blots were blocked and incubated overnight at 4 °C with antibodies against CASP6 (rabbit, 1:1000, Cell Signaling) and GAPDH (mouse, 1:20000, Millipore). .. The intensity of the selected bands was analyzed using NIH ImageJ software.

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000). .. Gel- and blot- band densitometry measurements were performed on unsaturated images using ImageJ software (National Institutes of Health).

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT. .. Images were analysed using ImageJ software to assess band intensity and values expressed relative to the loading control.

Co-Immunoprecipitation Assay:

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
Article Snippet: .. The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
Article Snippet: .. Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. Proximity ligation assay (PLA) To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

shRNA:

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: Antibodies and genetic constructs Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. .. VSV-G and pFIV-34N were kind gifts from Todd Waldman, Georgetown University. siRNA and shRNA expression constructs were obtained from Open Biosystems.

Article Title: RGS7 is recurrently mutated in melanoma and promotes migration and invasion of human cancer cells
Article Snippet: Anti-FLAG (M2) (Sigma-Aldrich) (cat no. F7425), anti-GAPDH (Millipore) (cat no. MAB374), anti-cyclin D1 (Cell Signaling) (cat no. 2926) PfuUltra II Hotstart PCR Master Mix (Agilent Technologies, Santa Clara, CA), the pCDF1-MCS2-EF1-Puro vector (Systems Biosciences, Inc., Mountain View, CA), Lipofectamine 2000 (Life Technologies) and cycloheximide (Sigma-Aldrich) were used. .. VSV-G and pFIV-34N were kind gifts from Todd Waldman, Georgetown University. siRNA and shRNA expression constructs were obtained from Open Biosystems.

Radio Immunoprecipitation:

Article Title: In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport
Article Snippet: Extracts using radioimmunoprecipitation assay (RIPA) buffer or 8 M urea dissolved in water were prepared as described before ( ). .. Primary antibodies were directed against β-actin (mouse, 1:10,000; Sigma no. A5441), Bim C34C5 (rabbit, 1:5,000; Cell Signaling no. 2933), vimentin (rabbit, 1:1,000; Acris no. AP00289PU-N), NF-κB p65 (rabbit, 1:5,000; Cell Signaling no. 4764), cytokeratin-8 (1:20,.000; Acris no. BM5045P), RFX5 (rabbit, 1:1,000; Rockland no. 200-401-194), USF-1 (rabbit, 1:2,000; Santa Cruz no. sc-8983), Cyclin B1 (mouse, 1:2,000; Cell Signaling no. 4135), GAPDH (mouse, 1:10,000; Millipore no. MAB374) and α-tubulin (mouse, 1:10,000; Sigma no. T9026).

Proximity Ligation Assay:

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
Article Snippet: .. The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB
Article Snippet: .. Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit). .. Proximity ligation assay (PLA) To create our PLA probes a rat anti-D1R antibody (Sigma, D2944) was conjugated with a PLUS oligonucleotide (Duolink® In Situ Probemaker PLUS DUO92009, Sigma-Olink) and a rabbit anti-D2R (Millipore, AB5084P) antibody with a MINUS oligonucleotide (Duolink® In Situ Probemaker MINUS DUO92010, Sigma-Olink) following manufacturer's instructions.

Homogenization:

Article Title: Gene Expression Profiling of Cutaneous Injured and Non-Injured Nociceptors in SNI Animal Model of Neuropathic Pain
Article Snippet: Western Blot Protein samples were prepared from dorsal root ganglia by homogenization in a lysis buffer containing protease and phosphatase inhibitors (Sigma-Aldrich), as previously described . .. The blots were blocked and incubated overnight at 4 °C with antibodies against CASP6 (rabbit, 1:1000, Cell Signaling) and GAPDH (mouse, 1:20000, Millipore).

Fractionation:

Article Title: Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Article Snippet: Following subcellular fractionation, RNA was isolated from each fraction as described above. .. The nuclear fraction was verified with Lamin A/C or Emerin (1:2000; anti-Rabbit, AbCam) and the cytoplasmic fraction with Gapdh (1:10,000; anti-mouse, Sigma, MS, USA) using Western Blot.

Staining:

Article Title: Muscle-specific CRISPR/Cas9 dystrophin gene editing ameliorates pathophysiology in a mouse model for Duchenne muscular dystrophy
Article Snippet: Blots were blocked for 1 hour at room temperature in 5% non-fat dry milk before overnight incubation with antibodies raised against the C-terminal domain of dystrophin (Froehner Lab, Rabbit polyclonal, 1:10,000), anti-SpCas9 (Millipore, mouse monoclonal, 1:2,000), anti-HA (Roche, Rat monoclonal-HRP conjugated, 1:2,000) for detection of HA-tagged saCas9 and Gapdh (Sigma, Rabbit polyclonal, 1:100,000). .. Horseradish-peroxidase conjugated secondary antibody staining (1:50,000) was performed for 1 h at room temperature before signal development using Clarity Western ECL substrate (BioRad) and visualization using a Chemidoc MP imaging system (BioRad).

Article Title: Biallelic mutations in the gene encoding eEF1A2 cause seizures and sudden death in F0 mice
Article Snippet: Membranes were incubated in 7% acetic acid and 10% methanol for 15 minutes, washed 4 times for 5 minutes in dH2 O and incubated in SYPRO Ruby stain for 15 minutes. .. Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT.

Article Title: Fructose-1,6-bisphosphate and aldolase mediate glucose sensing by AMPK
Article Snippet: Rabbit anti-PKLR (cat. ab38240, 1:500 for IB), rat anti-LAMP2 (cat. ab13524; 1:120 for IF), mouse anti-LAMP1 [cat. ab25630, 1:120 for immunofluorescent staining (IF)], mouse anti-aldolase (cat. ab54770, 1:50 for IP), and goat anti-TPI (cat. ab28760, 1:1000 for IB) antibodies were purchased from Abcam. .. Glucose (cat. G7021), G3P (cat. G5251), DHAP (cat. D7137), 3PG (cat. P8877), 2PG (cat. 19710), PEP (cat. P7002), N-acetylglucosamine (cat. A3286), octyl β-D-glucopyranoside (ODG, cat. O8001) and SLO (cat. S5265) were purchased from Sigma.

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    GAPDH Glyceraldehyde 3 Phosphate Dehydrogenase EC 1 2 1 12 catalyzes the conversion of Glyceraldehyde 3 Phosphate GAP to 1 3 Bisphosphate Glycerate BPG and plays a key role in
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    Millipore lc3b ii gapdh
    Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses <t>GFP-LC3B.</t> Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and <t>GAPDH</t> in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p
    Lc3b Ii Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 79/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gapdh
    Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, <t>4e-BP1</t> and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to <t>GAPDH</t> and values are expressed relative to the control group.
    Anti Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 641 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore gapdh proteins
    NF45 regulates survivin and cyclin E expression downstream of XIAP and <t>cIAP1.</t> (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G 2 /M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and <t>GAPDH.</t> (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.
    Gapdh Proteins, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Autophagy caused by starvation does not require MAPK8/9 in immortalized MEFs. (a) RPS6KB1, p-Thr389 RPS6KB1, and TUBA expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation with EBSS containing 5mM glucose (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the average of WT control condition (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Expressing, Incubation, Transduction, Plasmid Preparation, Fluorescence, Microscopy

    Autophagy caused by MTOR inhibition does not require MAPK8/9 in immortalized MEFs. (a) The amount of RPS6KB1, p-Thr389 RPS6KB1, and TUBA (tubulin alpha) in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation without or with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation of the cells with 250 nM torin 1 (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) without or with 250 nM torin 1 in the absence or presence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control condition (first lane). The ‘Change in MAP1LC3B-II’ was calculated by subtracting MAP1LC3B-II:GAPDH (media+ CQ condition) from MAP1LC3B-II:GAPDH (torin 1+ CQ condition). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Autophagy caused by MTOR inhibition does not require MAPK8/9 in immortalized MEFs. (a) The amount of RPS6KB1, p-Thr389 RPS6KB1, and TUBA (tubulin alpha) in WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation without or with 250 nM torin 1 (2 or 4 h) was examined by immunoblot analysis. (b) WT and mapk8 −/- mapk9 −/- immortalized MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation of the cells with 250 nM torin 1 (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 30 µm. (c) LC3B and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression by WT and mapk8 −/- mapk9 −/- immortalized MEFs after incubation (2 h) without or with 250 nM torin 1 in the absence or presence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control condition (first lane). The ‘Change in MAP1LC3B-II’ was calculated by subtracting MAP1LC3B-II:GAPDH (media+ CQ condition) from MAP1LC3B-II:GAPDH (torin 1+ CQ condition). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Inhibition, Incubation, Transduction, Plasmid Preparation, Fluorescence, Microscopy, Expressing

    Autophagy caused by starvation does not require MAPK8/9 in primary MEFs. (a) (Z)-4-Hydroxytamoxifen-treated primary Rosa- Cre ERT (WT) MEFs and Rosa- Cre ERT Mapk8 LoxP/LoxP mapk9 −/- MEFs were examined by immunoblot analysis by probing with antibodies to MAPK8/9 and TUBA. (b) WT and mapk8 −/- mapk9 −/- primary MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 25 µm. (c) LC3B and GAPDH expression by WT and mapk8 −/- mapk9 −/- primary MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Autophagy caused by starvation does not require MAPK8/9 in primary MEFs. (a) (Z)-4-Hydroxytamoxifen-treated primary Rosa- Cre ERT (WT) MEFs and Rosa- Cre ERT Mapk8 LoxP/LoxP mapk9 −/- MEFs were examined by immunoblot analysis by probing with antibodies to MAPK8/9 and TUBA. (b) WT and mapk8 −/- mapk9 −/- primary MEFs were transduced with a lentivirus vector that expresses GFP-LC3B. Puncta formation following incubation with EBSS containing 5 mM glucose (2 and 4 h) was examined by fluorescence microscopy. Scale bar: 25 µm. (c) LC3B and GAPDH expression by WT and mapk8 −/- mapk9 −/- primary MEFs after incubation (2 h) in medium or with EBSS containing 5 mM glucose in the presence or absence of 25 µM chloroquine (CQ) was examined by immunoblot analysis. The LC3B-II:GAPDH ratios were normalized to the mean of WT control (first lane). The data presented represent the mean ± SEM; n = 3 independent experiments; *, p

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Transduction, Plasmid Preparation, Incubation, Fluorescence, Microscopy, Expressing

    Effect of starvation and MTOR inhibition on MAPK8/9 activation is not sufficient to cause autophagy. (a and b) MAPK8/9 activation in immortalized MEFs was examined by immunoblot analysis of p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH in cells after incubation (2 or 4 h) with EBSS containing 5 mM glucose (a) or 250 nM torin 1 (b). Lanes 1 and 2 represent positive and negative controls: lysates of WT MEFs exposed to 60 J/m 2 UV and mapk8 −/- mapk9 −/- MEFs, respectively. (c) WT and mapk8 −/- mapk9 −/- immortalized MEFs were exposed to UV radiation (60 J/m 2 ) and cell extracts were prepared at 45 min post-irradiation. The expression of LC3B, p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH was examined by immunoblot analysis.

    Journal: Autophagy

    Article Title: Role of the MAPK/cJun NH2-terminal kinase signaling pathway in starvation-induced autophagy

    doi: 10.1080/15548627.2018.1466013

    Figure Lengend Snippet: Effect of starvation and MTOR inhibition on MAPK8/9 activation is not sufficient to cause autophagy. (a and b) MAPK8/9 activation in immortalized MEFs was examined by immunoblot analysis of p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH in cells after incubation (2 or 4 h) with EBSS containing 5 mM glucose (a) or 250 nM torin 1 (b). Lanes 1 and 2 represent positive and negative controls: lysates of WT MEFs exposed to 60 J/m 2 UV and mapk8 −/- mapk9 −/- MEFs, respectively. (c) WT and mapk8 −/- mapk9 −/- immortalized MEFs were exposed to UV radiation (60 J/m 2 ) and cell extracts were prepared at 45 min post-irradiation. The expression of LC3B, p-Thr183/Tyr185 MAPK8/9, MAPK8/9, and GAPDH was examined by immunoblot analysis.

    Article Snippet: Autophagic flux was examined by measuring the LC3B-II:GAPDH ratio or LC3B-II:TUBA (tubulin alpha) ratio by immunoblot analysis, normalization of the data to the control condition (without autophagy induction or CQ), and calculation of the increased LC3B-II:GAPDH (or TUBA) ratio caused by treatment of the cells with 25 µM chloroquine diphosphate (CQ; Millipore Sigma, 25,745) to inhibit lysosomal protein degradation [ ].

    Techniques: Inhibition, Activation Assay, Incubation, Irradiation, Expressing

    Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, 4e-BP1 and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to GAPDH and values are expressed relative to the control group.

    Journal: International Journal of Molecular Sciences

    Article Title: Secreted Protein Acidic and Rich in Cysteine (SPARC) Enhances Cell Proliferation, Migration, and Epithelial Mesenchymal Transition, and SPARC Expression is Associated with Tumor Grade in Head and Neck Cancer

    doi: 10.3390/ijms18071556

    Figure Lengend Snippet: Investigation of SPARC treatment-induced signaling pathways by Western blot: Detroit 562 cells were treated with SPARC for 1.5 h and the protein extract was subjected to Western blot assay. ( A ) Phosphorylation status of ribosomal s6 kinase1 (RSK1), mitogen and stress-activated protein kinase (MSK) , and extracellular signal-regulated kinase (ERK); ( B ) phosphorylation status of p70S6K, AKT, 4e-BP1 and ( C ) investigation of the effect of ERK, RSK, and AKT inhibitor. Band intensity was normalized to GAPDH and values are expressed relative to the control group.

    Article Snippet: The information and dilution fold is shown: anti-N -cadherin (1:1000, cat. no. 2167184, Millipore), anti-E-cadherin (1:1000, cat. no. 610182, BD Biosciences, San Jose, CA, USA), anti-Zo-1 (1:1000, cat. no. 5406S, Cell Signaling, Beverly, MA, USA), anti-Slug (1:1000, cat. no. 9585, Cell Signaling), anti-Snail (1:1000, cat. no.3879, Cell Signaling), anti-Twist (1:1000, cat. no. 49254, Abcam, Cambridge, UK), anti-phospho-ERK 1/2 (1:1000, cat. no. 4370S, Cell Signaling), anti-ERK 1/2 (1:1000, cat. no. 4695S, Cell Signaling), anti-phospho AKT Ser473 (1:1000, cat. no. 4060S, Cell Signaling), anti-phospho AKT Thr308 (1:1000, cat. no. 2965S, Cell Signaling), anti-phospho AKT (1:1000, cat. no. 9272S, Cell Signaling), anti-phospho Msk1 (1:1000, cat. no. 9591S, Cell Signaling), anti-Msk1 (1:1000, cat. no. 3489S, Cell Signaling), anti-phospho Rsk1 (1:1000, cat. no. 9335S, Cell Signaling), anti-Rsk1 (1:1000, cat. no. 8408S, Cell Signaling), anti-phospho p70 S6 (1:1000, cat. no. 9204, Cell Signaling), anti-p70 S6 (1:1000, cat. no. 9202, Cell Signaling), anti-phospho-4E-BP1 (1:1000, cat. no. 2855, Cell Signaling), anti-4E-BP1 (1:1000, cat. no. 9644, Cell Signaling), anti-GAPDH (1:5000, cat. no. MAB374, Millipore).

    Techniques: Western Blot

    Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p

    Journal: Frontiers in Pharmacology

    Article Title: Activation of Dopamine D1-D2 Receptor Complex Attenuates Cocaine Reward and Reinstatement of Cocaine-Seeking through Inhibition of DARPP-32, ERK, and ΔFosB

    doi: 10.3389/fphar.2017.00924

    Figure Lengend Snippet: Signaling pathways involved in D1-D2 heteromer modulation of cocaine-induced behaviors: DARPP-32, ERK and ΔFosB. (A) Representative immunoblots of pT34-DARPP-32 (top panel) or pT75-DARPP-32 (lower panel) obtained from NAc of rats conditioned with saline or cocaine (10 mg/kg, i.p.) and injected on the test day with saline or SKF 83959. Loading controls (GAPDH) are shown. Quantification of pT34- and pT75-DARPP-32 immunoblots is shown. (B) Representative immunoblot of pERK44/42 obtained from the NAc of rats conditioned as in (A) is shown. Quantification of pERK44/42 immunoblots obtained from all animals is shown. Results in (A,B) represent the mean ± SEM from 8 to 9 rats/condition. * p

    Article Snippet: Antibodies The primary antibodies used in the present study were raised against: Dopamine D1R (Sigma, D2944, rat); dopamine D2R for IHC and PLA (1st set) (Millipore; AB5084P; rabbit), dopamine D2R for co-IP (Alomone; Rabbit), dopamine D2R for second set PLA (Millipore, ABN 462), phospho-Thr75-DARPP-32 (Cell signaling, 2301s; rabbit), phospho-Thr34-DARPP-32 (Cell Signaling, 2304; rabbit), enkephalin (Millipore, MAB350, mouse), phospho-ERK1/2 (Sigma, E7028; rabbit), ΔFosB (Cell signaling, 14695S, rabbit), GAPDH (Millipore, rabbit).

    Techniques: Western Blot, Injection

    NF45 regulates survivin and cyclin E expression downstream of XIAP and cIAP1. (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G 2 /M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and GAPDH. (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleotide Composition of Cellular Internal Ribosome Entry Sites Defines Dependence on NF45 and Predicts a Posttranscriptional Mitotic Regulon

    doi: 10.1128/MCB.00546-12

    Figure Lengend Snippet: NF45 regulates survivin and cyclin E expression downstream of XIAP and cIAP1. (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G 2 /M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and GAPDH. (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.

    Article Snippet: Coimmunoprecipitations of cIAP1, XIAP, and GAPDH proteins were performed at 4°C overnight using protein G/protein A-agarose beads (EMD Chemicals) coated with, respectively, the antibodies anti-cIAP1 (R & D Systems, MN) at a 1:150 dilution, anti-GST-XIAP ( ) (Aegera) at a 1:150 dilution, and anti-GAPDH (clone 6C5; Advanced ImmunoChemical, CA) at a 1:250 dilution.

    Techniques: Expressing, Immunofluorescence, Staining, Flow Cytometry, Cytometry, Western Blot, Over Expression, Transfection, Plasmid Preparation

    NF45 regulates XIAP translation through interaction with its IRES. (A) Western blots of endogenous XIAP and cIAP1 protein expression in d5 cells relative to that in c cells. The blots and densitometry analyses are representative of at least three experiments. (B) NF45 reexpression in NF45-deficient HeLa cells rescues XIAP protein expression. The HeLa cells were transfected with 50 nM control nontargeting siRNA or NF45 siRNA (d5 siRNA) for 48 h, followed by the overexpression of GFP or GFP-NF45R for an additional 48 h. The cell extracts were analyzed by Western blotting for NF45, XIAP, cIAP1, and GAPDH expression, and protein expression was quantified. (C) De novo protein expression of cIAP1 and XIAP in NF45-deficient cells. c cells were transfected with 50 nM control or NF45 siRNA and were pulse-labeled with [ 35 S]methionine. 35 S-labeled and Coomassie blue-stained total protein, as well as specific cIAP1, XIAP, and GAPDH immunoprecipitates, are shown. (D) NF45 interacts specifically with the XIAP IRES. NF45 and PABP Western blotting of an RNA affinity chromatography preparation was performed using the XIAP IRES or hemoglobin RNA.

    Journal: Molecular and Cellular Biology

    Article Title: Nucleotide Composition of Cellular Internal Ribosome Entry Sites Defines Dependence on NF45 and Predicts a Posttranscriptional Mitotic Regulon

    doi: 10.1128/MCB.00546-12

    Figure Lengend Snippet: NF45 regulates XIAP translation through interaction with its IRES. (A) Western blots of endogenous XIAP and cIAP1 protein expression in d5 cells relative to that in c cells. The blots and densitometry analyses are representative of at least three experiments. (B) NF45 reexpression in NF45-deficient HeLa cells rescues XIAP protein expression. The HeLa cells were transfected with 50 nM control nontargeting siRNA or NF45 siRNA (d5 siRNA) for 48 h, followed by the overexpression of GFP or GFP-NF45R for an additional 48 h. The cell extracts were analyzed by Western blotting for NF45, XIAP, cIAP1, and GAPDH expression, and protein expression was quantified. (C) De novo protein expression of cIAP1 and XIAP in NF45-deficient cells. c cells were transfected with 50 nM control or NF45 siRNA and were pulse-labeled with [ 35 S]methionine. 35 S-labeled and Coomassie blue-stained total protein, as well as specific cIAP1, XIAP, and GAPDH immunoprecipitates, are shown. (D) NF45 interacts specifically with the XIAP IRES. NF45 and PABP Western blotting of an RNA affinity chromatography preparation was performed using the XIAP IRES or hemoglobin RNA.

    Article Snippet: Coimmunoprecipitations of cIAP1, XIAP, and GAPDH proteins were performed at 4°C overnight using protein G/protein A-agarose beads (EMD Chemicals) coated with, respectively, the antibodies anti-cIAP1 (R & D Systems, MN) at a 1:150 dilution, anti-GST-XIAP ( ) (Aegera) at a 1:150 dilution, and anti-GAPDH (clone 6C5; Advanced ImmunoChemical, CA) at a 1:250 dilution.

    Techniques: Western Blot, Expressing, Transfection, Over Expression, Labeling, Staining, Affinity Chromatography