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
Gapdh, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gapdh/product/Millipore
Average 90 stars, based on 149 article reviews
Price from $9.99 to $1999.99
gapdh - by Bioz Stars, 2020-04
90/100 stars

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 "Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation"

Article Title: Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025108

Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.
Figure Legend Snippet: Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.

Techniques Used: Mouse Assay, Staining, Marker

4) Product Images from "Cardiac Sympathetic Denervation Suppresses Atrial Fibrillation and Blood Pressure in a Chronic Intermittent Hypoxia Rat Model of Obstructive Sleep Apnea"

Article Title: Cardiac Sympathetic Denervation Suppresses Atrial Fibrillation and Blood Pressure in a Chronic Intermittent Hypoxia Rat Model of Obstructive Sleep Apnea

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.118.010254

Representative western blot analysis shows the protein expression of Cx43 among 4 groups. A , The expression of Cx43 protein was detected by western blots. Cx43 is specifically downregulated after CIH and CSD treatment increases Cx43 expression. GAPDH was used as an internal control. B , Quantification of the total Cx43 protein expression of the 4 groups. CIH indicates chronic intermittent hypoxia; CIH+CSD, chronic intermittent hypoxia with cardiac sympathetic denervation group; CSD, cardiac sympathetic denervation; Cx43, connexin 43.
Figure Legend Snippet: Representative western blot analysis shows the protein expression of Cx43 among 4 groups. A , The expression of Cx43 protein was detected by western blots. Cx43 is specifically downregulated after CIH and CSD treatment increases Cx43 expression. GAPDH was used as an internal control. B , Quantification of the total Cx43 protein expression of the 4 groups. CIH indicates chronic intermittent hypoxia; CIH+CSD, chronic intermittent hypoxia with cardiac sympathetic denervation group; CSD, cardiac sympathetic denervation; Cx43, connexin 43.

Techniques Used: Western Blot, Expressing

5) Product Images from "Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva)"

Article Title: Intracellular emetic signaling cascades by which the selective neurokinin type 1 receptor (NK1R) agonist GR73632 evokes vomiting in the least shrew (Cryptotis parva)

Journal: Neurochemistry international

doi: 10.1016/j.neuint.2018.11.012

Involvement of LTCC in NK 1 R-dependent PKCα/βII-ERK1/2 activation. A. Representative Western blots for time-course of PKC isoforms activation in the least shrew brainstems collected at indicated time points after GR73632 (5 mg/kg, i.p.) administration. Phospho-PKCα/βII Thr638/641 (pPKCα/βII), phospho-PKCδ Tyr311 (pPKCδ) and GAPDH (loading control) were detected by Western blot. B. Quantitative analysis of Western blots shown in A. The ratios of pPKCα/βII to GAPDH were compared. All ratios were normalized to vehicle-treated control (0 min) values before analysis and expressed as fold change of control. * p
Figure Legend Snippet: Involvement of LTCC in NK 1 R-dependent PKCα/βII-ERK1/2 activation. A. Representative Western blots for time-course of PKC isoforms activation in the least shrew brainstems collected at indicated time points after GR73632 (5 mg/kg, i.p.) administration. Phospho-PKCα/βII Thr638/641 (pPKCα/βII), phospho-PKCδ Tyr311 (pPKCδ) and GAPDH (loading control) were detected by Western blot. B. Quantitative analysis of Western blots shown in A. The ratios of pPKCα/βII to GAPDH were compared. All ratios were normalized to vehicle-treated control (0 min) values before analysis and expressed as fold change of control. * p

Techniques Used: Activation Assay, Western Blot

6) Product Images from "Pioglitazone Modulates the Vascular Contractility in Hypertension by Interference with ET-1 Pathway"

Article Title: Pioglitazone Modulates the Vascular Contractility in Hypertension by Interference with ET-1 Pathway

Journal: Scientific Reports

doi: 10.1038/s41598-019-52839-6

Contribution of NFκB to the ET-1-induced oxidative stress and COX-2 expression in VSMC from hypertensive rats. Effect of lactacystin (Lac, 10 µM) on NOX-1 mRNA levels ( a ), NADPH oxidase activity ( b ), COX-2 protein expression ( c ) and mRNA levels ( d ) induced by ET-1 (0.1 µM, 1 h). ( e ) Effect of ET-1 (0.1 µM, 45 min) on nuclear p65 NFκB protein expression in VSMC from SHR; a representative blot of the cytosolic (Cy) and nuclear (Nu) expression is shown below; TATA-binding protein (TBP) and GAPDH (after reblotting) cytosolic and nuclear expressions are also shown to guarantee the successful cellular fractioning. ( f ) Representative photomicrographs of p65 NFκB immunofluorescence (red) in VSMC from SHR in control and after incubation with ET-1 (0.1 µM, 45 min, n = 7). Negative controls without primary or secondary antibody are also shown. Bar scale represents 50 µm. Statistical analysis by Student’s t-test; * P
Figure Legend Snippet: Contribution of NFκB to the ET-1-induced oxidative stress and COX-2 expression in VSMC from hypertensive rats. Effect of lactacystin (Lac, 10 µM) on NOX-1 mRNA levels ( a ), NADPH oxidase activity ( b ), COX-2 protein expression ( c ) and mRNA levels ( d ) induced by ET-1 (0.1 µM, 1 h). ( e ) Effect of ET-1 (0.1 µM, 45 min) on nuclear p65 NFκB protein expression in VSMC from SHR; a representative blot of the cytosolic (Cy) and nuclear (Nu) expression is shown below; TATA-binding protein (TBP) and GAPDH (after reblotting) cytosolic and nuclear expressions are also shown to guarantee the successful cellular fractioning. ( f ) Representative photomicrographs of p65 NFκB immunofluorescence (red) in VSMC from SHR in control and after incubation with ET-1 (0.1 µM, 45 min, n = 7). Negative controls without primary or secondary antibody are also shown. Bar scale represents 50 µm. Statistical analysis by Student’s t-test; * P

Techniques Used: Expressing, Activity Assay, Binding Assay, Immunofluorescence, Incubation

7) Product Images from "Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation"

Article Title: Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025108

Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.
Figure Legend Snippet: Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.

Techniques Used: Mouse Assay, Staining, Marker

Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.
Figure Legend Snippet: Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.

Techniques Used: Mouse Assay, Staining, Marker

8) Product Images from "BMPR1a and BMPR1b Signaling Exert Opposing Effects on Gliosis after Spinal Cord Injury"

Article Title: BMPR1a and BMPR1b Signaling Exert Opposing Effects on Gliosis after Spinal Cord Injury

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.4459-09.2010

BMPR1a signaling affects GFAP levels independent of the canonical SMAD signaling pathway. A , Western blots for GFAP protein in injured cords from WT and BMPR1a CKO mice at 6 d post-SCI. The blot was stripped and reprobed for GAPDH (loading control).
Figure Legend Snippet: BMPR1a signaling affects GFAP levels independent of the canonical SMAD signaling pathway. A , Western blots for GFAP protein in injured cords from WT and BMPR1a CKO mice at 6 d post-SCI. The blot was stripped and reprobed for GAPDH (loading control).

Techniques Used: Western Blot, Mouse Assay

9) Product Images from "PTEN suppresses axon outgrowth by down-regulating the level of detyrosinated microtubules"

Article Title: PTEN suppresses axon outgrowth by down-regulating the level of detyrosinated microtubules

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193257

PI(3,4,5)P3 analog stimulates detyrosination of microtubules. [A] NIH/3T3 cells were siRNA depleted for GAPDH (control) or PTEN and serum depleted overnight. Cell lysates were analyzed by western blotting against PTEN, phospho-FAK (Y397), total FAK, phospho-PDK1, phospho-Akt (Ser473), total Akt, phospho-GSK3β, total GSK3β and β-Actin. Representative images of 3 independent experiments are shown. [B, C] NIH/3T3 cells were siRNA depleted against GAPDH or PTEN, treated with FAK inhibitors (PF-562271[1 μM], PF-573228 [10 μM]) as indicated and seeded on fibronectin-coated coverslips for 2 h. [B] Representative images of cells immunostained against, detyrosinated tubulin (red), total tubulin (green) and DNA (blue). [C] Percentage of detyrosinated microtubules positive cells. Error bar = StdDev, N = 4 (total of > 150 cells each). [D, E] NIH/3T3 cells were serum depleted for 2 days and treated with LPA [10 μM] (positive control) or PIP4-AM as indicated. [D] Representative image of cells immunostained against detyrosinated tubulin (deTyr, red), total tubulin (green) and DNA (blue). [E] Percentage of deTyr positive cells. Error bar = StdDev, N = 3 (total of > 150 cells each). Scale bars, 10 μm, ** p
Figure Legend Snippet: PI(3,4,5)P3 analog stimulates detyrosination of microtubules. [A] NIH/3T3 cells were siRNA depleted for GAPDH (control) or PTEN and serum depleted overnight. Cell lysates were analyzed by western blotting against PTEN, phospho-FAK (Y397), total FAK, phospho-PDK1, phospho-Akt (Ser473), total Akt, phospho-GSK3β, total GSK3β and β-Actin. Representative images of 3 independent experiments are shown. [B, C] NIH/3T3 cells were siRNA depleted against GAPDH or PTEN, treated with FAK inhibitors (PF-562271[1 μM], PF-573228 [10 μM]) as indicated and seeded on fibronectin-coated coverslips for 2 h. [B] Representative images of cells immunostained against, detyrosinated tubulin (red), total tubulin (green) and DNA (blue). [C] Percentage of detyrosinated microtubules positive cells. Error bar = StdDev, N = 4 (total of > 150 cells each). [D, E] NIH/3T3 cells were serum depleted for 2 days and treated with LPA [10 μM] (positive control) or PIP4-AM as indicated. [D] Representative image of cells immunostained against detyrosinated tubulin (deTyr, red), total tubulin (green) and DNA (blue). [E] Percentage of deTyr positive cells. Error bar = StdDev, N = 3 (total of > 150 cells each). Scale bars, 10 μm, ** p

Techniques Used: Western Blot, Positive Control

10) 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

11) Product Images from "Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation"

Article Title: Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0025108

Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.
Figure Legend Snippet: Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.

Techniques Used: Mouse Assay, Staining, Marker

Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.
Figure Legend Snippet: Adult WT and Type III Nrg1 +/− animals have equivalent numbers of sensory neurons and sensory cutaneous projections. (A) Representative sections through L4/L5 DRG from adult WT and Type III Nrg1 +/− mice were stained with a pan-sensory marker. Total cells staining positive for this marker tallied from at least 5 sections evenly spaced throughout the DRG were counted and averaged for each animal. Genotype averages were compared with a Student's t-test (n = 3 animals per genotype). There was no statistically significant difference between genotypes. (B) Galabrous hindpaw skin samples from WT and Type III Nrg1 +/− mice were assessed for total TrkA, Ret and TRPV1 protein using immunoblot. (C) The intensity of the TrkA, Ret and TRPV1 bands were quantified and normalized to GAPDH to control for equal protein loading. The values were expressed as a fold change from the average WT value and genotype averages were compared with a Student's t-test (for TrkA and Ret: WT n = 13 paws from 10 animals, Type III Nrg1 +/− n = 13 paws from 9 animals; for TRPV1: WT n = 9 paws from 5 animals, Type III Nrg1 +/− n = 8 paws from 5 animals). There was no statistically significant difference between the genotypes.

Techniques Used: Mouse Assay, Staining, Marker

12) 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

13) 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

14) Product Images from "LIN28 Expression in Rat Spinal Cord After Injury"

Article Title: LIN28 Expression in Rat Spinal Cord After Injury

Journal: Neurochemical Research

doi: 10.1007/s11064-014-1278-2

The expression and correlation of LIN28 and NF-κB signaling pathway during LPS induced astrocytes activation. Astrocytes were treated with LPS (1 μg/ml) for indicated times. INOS, p65, p50, IκBα, p-IκBα, p-p65, LIN28, and GAPDH expression was determined by Western blot analysis ( a ). Quantification of INOS, p65, p50, IκBα, p-IκBα, p-p65, and LIN28 protein levels. (n = 3, * p
Figure Legend Snippet: The expression and correlation of LIN28 and NF-κB signaling pathway during LPS induced astrocytes activation. Astrocytes were treated with LPS (1 μg/ml) for indicated times. INOS, p65, p50, IκBα, p-IκBα, p-p65, LIN28, and GAPDH expression was determined by Western blot analysis ( a ). Quantification of INOS, p65, p50, IκBα, p-IκBα, p-p65, and LIN28 protein levels. (n = 3, * p

Techniques Used: Expressing, Activation Assay, Western Blot

Time-dependent expression of LIN28 protein in rat spinal cord after SCI. Spinal cord tissues from rats at various survival times after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with LIN28 and GAPDH are shown above ( a ). The bar chart below demonstrates the ratio of LIN28 to GAPDH for each time point ( b ). Data are mean ± SEM (n = 3; * p
Figure Legend Snippet: Time-dependent expression of LIN28 protein in rat spinal cord after SCI. Spinal cord tissues from rats at various survival times after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with LIN28 and GAPDH are shown above ( a ). The bar chart below demonstrates the ratio of LIN28 to GAPDH for each time point ( b ). Data are mean ± SEM (n = 3; * p

Techniques Used: Expressing, Western Blot

Dose-response effects’ between the SCI and LIN28 expression. Spinal cord tissues from rats at various degrees of contusion induced by different heights (0, 6.25, 12.5, 25, 50, 75, 100, and 125 mm) at day 1 after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with LIN28 and GAPDH are shown above ( a ). The bar chart below demonstrates the ratio of LIN28 to GAPDH for each height point ( b ). Data are mean ± SEM (n = 3; * p
Figure Legend Snippet: Dose-response effects’ between the SCI and LIN28 expression. Spinal cord tissues from rats at various degrees of contusion induced by different heights (0, 6.25, 12.5, 25, 50, 75, 100, and 125 mm) at day 1 after SCI were homogenized and subjected to immunoblot analysis. Sample immunoblots probed with LIN28 and GAPDH are shown above ( a ). The bar chart below demonstrates the ratio of LIN28 to GAPDH for each height point ( b ). Data are mean ± SEM (n = 3; * p

Techniques Used: Expressing, Western Blot

15) Product Images from "Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology"

Article Title: Whole Genome Expression Analysis in a Mouse Model of Tauopathy Identifies MECP2 as a Possible Regulator of Tau Pathology

Journal: Frontiers in Molecular Neuroscience

doi: 10.3389/fnmol.2017.00069

MECP2 phosphorylation is up-regulated in 12-month-old hTau MaptKO ( Duke ) mice and in human AD brain. (A,B) Western blot analysis showing pMECP2/GAPDH ratio significantly higher [ ∗ p
Figure Legend Snippet: MECP2 phosphorylation is up-regulated in 12-month-old hTau MaptKO ( Duke ) mice and in human AD brain. (A,B) Western blot analysis showing pMECP2/GAPDH ratio significantly higher [ ∗ p

Techniques Used: Mouse Assay, Western Blot

16) Product Images from "Role of Adipose Tissue Nutrient/Vitamin Metabolism in Physiological and Altered Metabolic Settings: Insulin-like growth factor-II in adipocyte regulation: depot-specific actions suggest a potential role limiting excess visceral adiposity"

Article Title: Role of Adipose Tissue Nutrient/Vitamin Metabolism in Physiological and Altered Metabolic Settings: Insulin-like growth factor-II in adipocyte regulation: depot-specific actions suggest a potential role limiting excess visceral adiposity

Journal: American Journal of Physiology - Endocrinology and Metabolism

doi: 10.1152/ajpendo.00409.2017

Effect of IGF-II treatment on GLUT 4 and glucose uptake in differentiated adipocytes. A : relative mRNA expression levels of GLUT4 in subcutaneous and visceral adipocytes measured by qPCR and normalized to GAPDH reference gene. B : Western blot showing GLUT4 protein abundance; GAPDH used a loading control. C : densitometry analysis of B indicating a reduction in GLUT4 protein abundance in visceral adipocytes. D : [ 3 H]2-Deoxy glucose uptake following IGF-II treatment in visceral and subcutaneous adipocytes. Data expressed as the means ± SE of duplicate runs for qPCR; each Western blot densitometry is representative of experiments performed in triplicate from three individual biopsies ( n = 3). Statistical analysis performed using one-way ANOVA (* P
Figure Legend Snippet: Effect of IGF-II treatment on GLUT 4 and glucose uptake in differentiated adipocytes. A : relative mRNA expression levels of GLUT4 in subcutaneous and visceral adipocytes measured by qPCR and normalized to GAPDH reference gene. B : Western blot showing GLUT4 protein abundance; GAPDH used a loading control. C : densitometry analysis of B indicating a reduction in GLUT4 protein abundance in visceral adipocytes. D : [ 3 H]2-Deoxy glucose uptake following IGF-II treatment in visceral and subcutaneous adipocytes. Data expressed as the means ± SE of duplicate runs for qPCR; each Western blot densitometry is representative of experiments performed in triplicate from three individual biopsies ( n = 3). Statistical analysis performed using one-way ANOVA (* P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot

Characterization of subcutaneous and visceral preadipocyte fat biopsies from prepubertal children. A : photomicrographs of human subcutaneous and visceral preadipocytes ( day 0 ) displaying a fibroblastic morphology and differentiated adipocytes ( day 14 ) stained with Oil Red O. Magnification at (×10). B : quantitative absorbance analysis of Oil Red O staining showing a significant increase in fat deposition in mature adipocytes in comparison with preadipocytes. C : Western blotting of the differentiation markers (PPAR-γ, adiponectin) in subcutaneous and visceral preadipocytes and in differentiated adipocytes ( day 14 ). β-Actin was used as a loading control. D : quantitative densitometry analysis of Western blot indicating an increase in differentiation marker expression. E : relative mRNA expression of differentiation markers using qPCR, indicating an increase in differentiation. GAPDH used as reference gene. Data expressed as the means ± SE of duplicate runs for absorbance analysis and qPCR; each Western blot densitometry is representative of experiments performed in triplicate from four individual biopsies ( n = 4). Statistical analysis performed using one-way ANOVA (** P
Figure Legend Snippet: Characterization of subcutaneous and visceral preadipocyte fat biopsies from prepubertal children. A : photomicrographs of human subcutaneous and visceral preadipocytes ( day 0 ) displaying a fibroblastic morphology and differentiated adipocytes ( day 14 ) stained with Oil Red O. Magnification at (×10). B : quantitative absorbance analysis of Oil Red O staining showing a significant increase in fat deposition in mature adipocytes in comparison with preadipocytes. C : Western blotting of the differentiation markers (PPAR-γ, adiponectin) in subcutaneous and visceral preadipocytes and in differentiated adipocytes ( day 14 ). β-Actin was used as a loading control. D : quantitative densitometry analysis of Western blot indicating an increase in differentiation marker expression. E : relative mRNA expression of differentiation markers using qPCR, indicating an increase in differentiation. GAPDH used as reference gene. Data expressed as the means ± SE of duplicate runs for absorbance analysis and qPCR; each Western blot densitometry is representative of experiments performed in triplicate from four individual biopsies ( n = 4). Statistical analysis performed using one-way ANOVA (** P

Techniques Used: Staining, Western Blot, Marker, Expressing, Real-time Polymerase Chain Reaction

17) Product Images from "Hyperosmotic stress stimulates autophagy via polycystin-2"

Article Title: Hyperosmotic stress stimulates autophagy via polycystin-2

Journal: Oncotarget

doi: 10.18632/oncotarget.18995

Pro-survival role of autophagy in cells subjected to hyperosmotic stress HeLa cells were transfected with an unrelated control siRNA (siUNR) or specific siRNAs against PC2 and Beclin 1. 48 h later, cells were exposed to sorbitol (200 mOsm) at the indicated times. Pro-caspase-3, Beclin 1 and caspase-3 levels were evaluated by Western blot analysis. GAPDH levels were used as a loading control. Representative gels are shown in A. Quantification of gel bands is shown in B. (mean ± SEM, n = 3, * p
Figure Legend Snippet: Pro-survival role of autophagy in cells subjected to hyperosmotic stress HeLa cells were transfected with an unrelated control siRNA (siUNR) or specific siRNAs against PC2 and Beclin 1. 48 h later, cells were exposed to sorbitol (200 mOsm) at the indicated times. Pro-caspase-3, Beclin 1 and caspase-3 levels were evaluated by Western blot analysis. GAPDH levels were used as a loading control. Representative gels are shown in A. Quantification of gel bands is shown in B. (mean ± SEM, n = 3, * p

Techniques Used: Transfection, Western Blot

18) Product Images from "PTEN suppresses axon outgrowth by down-regulating the level of detyrosinated microtubules"

Article Title: PTEN suppresses axon outgrowth by down-regulating the level of detyrosinated microtubules

Journal: PLoS ONE

doi: 10.1371/journal.pone.0193257

PI(3,4,5)P3 analog stimulates detyrosination of microtubules. [A] NIH/3T3 cells were siRNA depleted for GAPDH (control) or PTEN and serum depleted overnight. Cell lysates were analyzed by western blotting against PTEN, phospho-FAK (Y397), total FAK, phospho-PDK1, phospho-Akt (Ser473), total Akt, phospho-GSK3β, total GSK3β and β-Actin. Representative images of 3 independent experiments are shown. [B, C] NIH/3T3 cells were siRNA depleted against GAPDH or PTEN, treated with FAK inhibitors (PF-562271[1 μM], PF-573228 [10 μM]) as indicated and seeded on fibronectin-coated coverslips for 2 h. [B] Representative images of cells immunostained against, detyrosinated tubulin (red), total tubulin (green) and DNA (blue). [C] Percentage of detyrosinated microtubules positive cells. Error bar = StdDev, N = 4 (total of > 150 cells each). [D, E] NIH/3T3 cells were serum depleted for 2 days and treated with LPA [10 μM] (positive control) or PIP4-AM as indicated. [D] Representative image of cells immunostained against detyrosinated tubulin (deTyr, red), total tubulin (green) and DNA (blue). [E] Percentage of deTyr positive cells. Error bar = StdDev, N = 3 (total of > 150 cells each). Scale bars, 10 μm, ** p
Figure Legend Snippet: PI(3,4,5)P3 analog stimulates detyrosination of microtubules. [A] NIH/3T3 cells were siRNA depleted for GAPDH (control) or PTEN and serum depleted overnight. Cell lysates were analyzed by western blotting against PTEN, phospho-FAK (Y397), total FAK, phospho-PDK1, phospho-Akt (Ser473), total Akt, phospho-GSK3β, total GSK3β and β-Actin. Representative images of 3 independent experiments are shown. [B, C] NIH/3T3 cells were siRNA depleted against GAPDH or PTEN, treated with FAK inhibitors (PF-562271[1 μM], PF-573228 [10 μM]) as indicated and seeded on fibronectin-coated coverslips for 2 h. [B] Representative images of cells immunostained against, detyrosinated tubulin (red), total tubulin (green) and DNA (blue). [C] Percentage of detyrosinated microtubules positive cells. Error bar = StdDev, N = 4 (total of > 150 cells each). [D, E] NIH/3T3 cells were serum depleted for 2 days and treated with LPA [10 μM] (positive control) or PIP4-AM as indicated. [D] Representative image of cells immunostained against detyrosinated tubulin (deTyr, red), total tubulin (green) and DNA (blue). [E] Percentage of deTyr positive cells. Error bar = StdDev, N = 3 (total of > 150 cells each). Scale bars, 10 μm, ** p

Techniques Used: Western Blot, Positive Control

19) Product Images from "BTEB2 Prevents Neuronal Apoptosis via Promoting Bad Phosphorylation in Rat Intracerebral Hemorrhage Model"

Article Title: BTEB2 Prevents Neuronal Apoptosis via Promoting Bad Phosphorylation in Rat Intracerebral Hemorrhage Model

Journal: Journal of Molecular Neuroscience

doi: 10.1007/s12031-014-0305-8

Association of BTEB2 with neuronal expressed active caspase-3 after ICH. The expression of active caspase-3 increased and peaked at day 3 after ICH, GAPDH was used to confirm that equal amount of protein was run on gel ( a ).Quantification graphs (relative optical density) of the intensity of staining of active caspase-3 and GAPDH at each time points ( b ). Negative correlation between BTEB2 protein expression and active caspase-3 protein expression after ICH ( c ). The data are mean ± SEM ( p
Figure Legend Snippet: Association of BTEB2 with neuronal expressed active caspase-3 after ICH. The expression of active caspase-3 increased and peaked at day 3 after ICH, GAPDH was used to confirm that equal amount of protein was run on gel ( a ).Quantification graphs (relative optical density) of the intensity of staining of active caspase-3 and GAPDH at each time points ( b ). Negative correlation between BTEB2 protein expression and active caspase-3 protein expression after ICH ( c ). The data are mean ± SEM ( p

Techniques Used: Expressing, Staining

BTEB2 prevents neuronal apoptosis through Bad phosphorylation. Western blot showed siRNA knocked down BTEB2 expression in PC12 cells ( a ). Knocking down BTEB2 induced increasing levels of active caspase-3 and reduced the levels of P-ser112-bad and P-ser136-bad in hemin-treated PC12 cells ( c ). The bar chart indicates the density of active caspase-3/BTEB2/P-ser112-Bad/P-ser136-Bad versus GAPDH ( b , d ). Data are presented as means ± SEM (* p
Figure Legend Snippet: BTEB2 prevents neuronal apoptosis through Bad phosphorylation. Western blot showed siRNA knocked down BTEB2 expression in PC12 cells ( a ). Knocking down BTEB2 induced increasing levels of active caspase-3 and reduced the levels of P-ser112-bad and P-ser136-bad in hemin-treated PC12 cells ( c ). The bar chart indicates the density of active caspase-3/BTEB2/P-ser112-Bad/P-ser136-Bad versus GAPDH ( b , d ). Data are presented as means ± SEM (* p

Techniques Used: Western Blot, Expressing

BTEB2 protein expression following ICH. Western blot was performed to study the protein level of BTEB2 surrounding the hematoma at different survival times ( a ). Quantification graphs of the intensity of staining of BTEB2 to GAPDH at each time point ( b ). Data are presented as mean ± SEM ( n = 3, * p
Figure Legend Snippet: BTEB2 protein expression following ICH. Western blot was performed to study the protein level of BTEB2 surrounding the hematoma at different survival times ( a ). Quantification graphs of the intensity of staining of BTEB2 to GAPDH at each time point ( b ). Data are presented as mean ± SEM ( n = 3, * p

Techniques Used: Expressing, Western Blot, Staining

20) Product Images from "The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts"

Article Title: The Retinoblastoma Tumor Suppressor Transcriptionally Represses Pak1 in Osteoblasts

Journal: PLoS ONE

doi: 10.1371/journal.pone.0142406

Rb interacts with the Pak1 promoter in complex with E2F. (A) ChIP assays showing interaction of Rb with the Pak1 promoter. The Pak1 promoter primer set used (-201/+250) is the same as illustrated in Fig 3B . The osteocalcin (OC) promoter was used as a positive control since Rb has been shown to physically interact with this promoter [ 4 ]. Negative controls were performed either by omitting DNA from the PCR reaction (no DNA or ND) or by immunoprecipitating with an irrelevant antibody against GAPDH. L, DNA ladder; In, PCR reactions done with input DNA not subjected to immunoprecipitation; IP, samples immunoprecipitated with anti-Rb antibody. (B) ChIP assays showing that Rb binds to the Pak1 promoter in complex with E2F1. ChIP assays in which the immunoprecipitation was done with anti-E2F1 antibody is labeled as IP-E2F. ChIP assays in which an immunoprecipitation done with an anti-Rb antibody followed by a second round of immunoprecipitation with an anti-E2F1 antibody is labeled as IP-Rb-E2F. PCR using primers against the Pak1 promoter resulted in a product, indicating interaction of the Rb-E2F complex to the Pak1 promoter. L, DNA ladder; In, PCR done with non-immunoprecipitated input DNA; ND, negative control with no DNA added to the PCR reaction.
Figure Legend Snippet: Rb interacts with the Pak1 promoter in complex with E2F. (A) ChIP assays showing interaction of Rb with the Pak1 promoter. The Pak1 promoter primer set used (-201/+250) is the same as illustrated in Fig 3B . The osteocalcin (OC) promoter was used as a positive control since Rb has been shown to physically interact with this promoter [ 4 ]. Negative controls were performed either by omitting DNA from the PCR reaction (no DNA or ND) or by immunoprecipitating with an irrelevant antibody against GAPDH. L, DNA ladder; In, PCR reactions done with input DNA not subjected to immunoprecipitation; IP, samples immunoprecipitated with anti-Rb antibody. (B) ChIP assays showing that Rb binds to the Pak1 promoter in complex with E2F1. ChIP assays in which the immunoprecipitation was done with anti-E2F1 antibody is labeled as IP-E2F. ChIP assays in which an immunoprecipitation done with an anti-Rb antibody followed by a second round of immunoprecipitation with an anti-E2F1 antibody is labeled as IP-Rb-E2F. PCR using primers against the Pak1 promoter resulted in a product, indicating interaction of the Rb-E2F complex to the Pak1 promoter. L, DNA ladder; In, PCR done with non-immunoprecipitated input DNA; ND, negative control with no DNA added to the PCR reaction.

Techniques Used: Chromatin Immunoprecipitation, Positive Control, Polymerase Chain Reaction, Immunoprecipitation, Labeling, Negative Control

21) 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

22) 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

23) 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

24) 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

25) 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

MTT Assay:

Article Title: Kanglaite sensitizes colorectal cancer cells to Taxol via NF-κΒ inhibition and connexin 43 upregulation
Article Snippet: .. Rabbit anti-NF-κΒ p65 antibody, rabbit anti-IKKα antibody, rabbit anti-IκΒα antibody, rabbit anti-caspase-8 antibody, rabbit anti-GAPDH antibody, rabbit anti-Poly[ADP-ribose] Polymerase (PARP) antibody, rabbit anti-cyclin B1 antibody, rabbit anti-survivin antibody, rabbit anti-caspase-3 antibody, mouse anti-α-tubulin antibody, rabbit anti-β-actin antibody, goat anti-rabbit IgG-peroxidase, goat anti-mouse IgG-peroxidase, goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC, DAPI, methyl thiazolyl tetrazolium (MTT), and Taxol were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. Immobilon membranes were purchased from Merck Millipore (Bedford, MA, USA).

Centrifugation:

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: The beads were precipitated by centrifugation at 6 000 r.p.m. for 1 min, and immunoprecipitates were washed three times in RIPA buffer and subjected to western blotting analysis. .. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody.

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation
Article Snippet: The lysates (300 μ l or 150 μ l for each 6-cm dish or well of a six-well plate, respectively) were transferred into 1.5-ml Eppendorf tubes, which were placed on a rotator at 4 °C for 30 min before centrifugation at 16 300 × g for 10 min at 4 °C. .. Equal loading was confirmed by probing the blots with antibodies against GAPDH (rabbit polyclonal, 1 : 5000) or β -actin (mouse monoclonal, 1 : 10 000), both from Sigma, Dorset, UK.

Neutralization:

Article Title: Mutation S110L of H1N1 Influenza Virus Hemagglutinin: A Potent Determinant of Attenuation in the Mouse Model
Article Snippet: Antibodies rabbit anti-GAPDH, (Sigma), rabbit anti-PB1 , rabbit anti-NP , and mouse anti-HA ( ) were used for western blot assays. .. Mouse anti-HA and rabbit anti-HA ( ) were used for neutralization assay.

BIA-KA:

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation
Article Snippet: Equal loading was confirmed by probing the blots with antibodies against GAPDH (rabbit polyclonal, 1 : 5000) or β -actin (mouse monoclonal, 1 : 10 000), both from Sigma, Dorset, UK. .. Equal loading was confirmed by probing the blots with antibodies against GAPDH (rabbit polyclonal, 1 : 5000) or β -actin (mouse monoclonal, 1 : 10 000), both from Sigma, Dorset, UK.

Article Title: Kanglaite sensitizes colorectal cancer cells to Taxol via NF-κΒ inhibition and connexin 43 upregulation
Article Snippet: Rabbit anti-NF-κΒ p65 antibody, rabbit anti-IKKα antibody, rabbit anti-IκΒα antibody, rabbit anti-caspase-8 antibody, rabbit anti-GAPDH antibody, rabbit anti-Poly[ADP-ribose] Polymerase (PARP) antibody, rabbit anti-cyclin B1 antibody, rabbit anti-survivin antibody, rabbit anti-caspase-3 antibody, mouse anti-α-tubulin antibody, rabbit anti-β-actin antibody, goat anti-rabbit IgG-peroxidase, goat anti-mouse IgG-peroxidase, goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC, DAPI, methyl thiazolyl tetrazolium (MTT), and Taxol were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. ECL Plus substrate, bicinchoninic acid (BCA) reagents, and RIPA lysis buffer were purchased from CWBio (Beijing, China).

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: BCA protein assay reagent (Pierce) was used to estimate the protein concentrations, and 30 μg of proteins was used to detect δ subunit signals in 10% SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes. .. The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ).

Western Blot:

Article Title: Expression and localization of VPAC1, the major receptor of vasoactive intestinal peptide along the length of the intestine
Article Snippet: Paragraph title: Western blot analysis. ... The membranes were blocked with 5% milk in phosphate-buffered saline (PBS) for 1 h, followed by incubation with anti-rabbit VPAC1 primary antibody at a dilution of 1 to 250 or anti-rabbit GAPDH antibody (Sigma-Aldrich, St. Louis, MO) at a ratio of 1 to 10,000 in 1% milk in PBS overnight at 4°C.

Article Title: Notch1 functions as a tumor suppressor in a model of K-ras-induced pancreatic ductal adenocarcinoma
Article Snippet: Paragraph title: Western blot analysis ... Primary antibodies: rabbit anti-Notch1 (1:500, Epitomics), rabbit anti-Notch2, 3 and 4 (all 1:200, Santa Cruz), mouse anti-β-catenin (1:1000, BD Biosciences), mouse anti-active β-catenin (1:1000, Millipore), rabbit anti-GAPDH (1:10,000, Sigma) and mouse anti-tubulin (1:8000, Sigma).

Article Title: Mutation S110L of H1N1 Influenza Virus Hemagglutinin: A Potent Determinant of Attenuation in the Mouse Model
Article Snippet: .. Antibodies rabbit anti-GAPDH, (Sigma), rabbit anti-PB1 , rabbit anti-NP , and mouse anti-HA ( ) were used for western blot assays. .. Mouse anti-HA and rabbit anti-HA ( ) were used for neutralization assay.

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: Paragraph title: Co-immunoprecipitation and western blotting ... After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody.

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation
Article Snippet: Paragraph title: Western blotting ... Equal loading was confirmed by probing the blots with antibodies against GAPDH (rabbit polyclonal, 1 : 5000) or β -actin (mouse monoclonal, 1 : 10 000), both from Sigma, Dorset, UK.

Article Title: Subchronic Administration and Combination Metabotropic Glutamate and GABAB Receptor Drug Therapy in Fragile X Syndrome
Article Snippet: Paragraph title: Western Blotting. ... The following antibodies were used: mouse anti-GABAB R1 (clone NR3A/49; 1:250; NeuroMab, University of California, Davis, CA/National Institutes of Health, Bethesda, MD); mouse anti-GABAB R2 (clone N81/2; 1:750; NeuroMab, University of California, Davis/National Institutes of Health); rabbit anti-mGluR5 (1:500; Millipore Bioscience Research Reagents, Temecula, CA); mouse anti-GAPDH antibody (1:40,000–1:100,000; Sigma-Aldrich); and a goat anti-mouse (The Jackson Laboratory, Bar Harbor, ME) or goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA) horseradish peroxidase-conjugated secondary antibody.

Article Title: Repeated Sigma-1 Receptor Antagonist MR309 Administration Modulates Central Neuropathic Pain Development After Spinal Cord Injury in Mice
Article Snippet: Paragraph title: Western Blotting ... To ensure equal protein loading, rabbit anti-GAPDH antibody (1:40,000, Sigma-Aldrich Química S.A.) was used as a loading control.

Article Title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Article Snippet: Paragraph title: Tissue lysates and Western blotting. ... Other primary antibodies used were rat anti-K8 antibody (Troma I, Developmental Studies Hybridoma Bank, University of Iowa), or rabbit-GAPDH antibody (Sigma and Cell Signaling).

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: Paragraph title: Western Blot Analysis ... The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ).

Article Title: Distinct Functions for Mammalian CLASP1 and -2 During Neurite and Axon Elongation
Article Snippet: Antibodies We used the following commercial primary antibodies (Abs): mouse anti-α-tubulin, mouse anti-β-tubulin, mouse anti-α-Tyrosinated-tubulin, mouse anti-α-Acetylated tubulin, mouse anti-β-actin, and rabbit anti-GAPDH (all from Sigma-Aldrich); mouse anti-βIII tubulin (Promega), mouse anti-α-detyrosinated (Glu)-tubulin (Abcam); mouse anti-GFP (Roche); rabbit anti-Akt-phosphoSer (Ser 473), rabbit anti-GSK3-α/β-phosphoSer (Ser 21/Ser9), rabbit anti-GSK3-α/β (Cell Signaling), rat anti-CLASP1 (#1A6) and -CLASP2 (#12H2, Absea antibodies). .. We also used previously described primary rabbit anti-CLASP1 [#402, for immunofluorescence (IF), and #2292 for western blot (WB)], and rabbit anti-CLASP2 (#2358, both for IF and WB; Akhmanova et al., ).

Transfection:

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: Co-immunoprecipitation and western blotting Extracts from E18.5 and postnatal day 0 (P0) mice or transfected cells were incubated on ice for 30 min in cold RIPA buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1% NP-40) with PMSF, phosphatase inhibitors and protease inhibitors. .. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody.

Blocking Assay:

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: .. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody. .. In vivo analysis To express shRNA or Ryk∆PDZ in CST axons, 0.6 μl of shRNA-EGFP-expressing lentivirus or Ryk∆PDZ-EGFP-expressing lentivirus was injected into the motor cortex at 0.8 mm from the midline, and the depth of the injection was 0.5 mm.

Article Title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Article Snippet: After 1 h of incubation in blocking buffer (1× PBS and 5% nonfat dry milk), the membranes were probed with affinity-purified rabbit anti-DRA antibody. .. Other primary antibodies used were rat anti-K8 antibody (Troma I, Developmental Studies Hybridoma Bank, University of Iowa), or rabbit-GAPDH antibody (Sigma and Cell Signaling).

Mouse Assay:

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: Co-immunoprecipitation and western blotting Extracts from E18.5 and postnatal day 0 (P0) mice or transfected cells were incubated on ice for 30 min in cold RIPA buffer (50 mm Tris-HCl, 150 mm NaCl, 1 mm EDTA, 1% NP-40) with PMSF, phosphatase inhibitors and protease inhibitors. .. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody.

Article Title: Subchronic Administration and Combination Metabotropic Glutamate and GABAB Receptor Drug Therapy in Fragile X Syndrome
Article Snippet: Immediately after seizure testing, mice were euthanized with an overdose of ketamine/xylazine, and the brains were removed and frozen on dry ice. .. The following antibodies were used: mouse anti-GABAB R1 (clone NR3A/49; 1:250; NeuroMab, University of California, Davis, CA/National Institutes of Health, Bethesda, MD); mouse anti-GABAB R2 (clone N81/2; 1:750; NeuroMab, University of California, Davis/National Institutes of Health); rabbit anti-mGluR5 (1:500; Millipore Bioscience Research Reagents, Temecula, CA); mouse anti-GAPDH antibody (1:40,000–1:100,000; Sigma-Aldrich); and a goat anti-mouse (The Jackson Laboratory, Bar Harbor, ME) or goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA) horseradish peroxidase-conjugated secondary antibody.

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: Pontine slices (300 μm thick) containing the LC area were obtained from 3- to 4-week-old mice with a vibratome sectioning system, and the pons was processed in radioimmune precipitation assay buffer (Sigma-Aldrich) with 1% protease inhibitor. .. The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ).

SDS-Gel:

Article Title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Article Snippet: Equal amounts (75 μg/sample) of tissue lysates were solubilized in SDS-gel loading buffer and boiled for 5 min. Lysates were run on 7.5–10% SDS-polyacrylamide gels and blotted to a nitrocellulose or polyvinylidene fluoride membrane after electrophoretic separation. .. Other primary antibodies used were rat anti-K8 antibody (Troma I, Developmental Studies Hybridoma Bank, University of Iowa), or rabbit-GAPDH antibody (Sigma and Cell Signaling).

Incubation:

Article Title: Expression and localization of VPAC1, the major receptor of vasoactive intestinal peptide along the length of the intestine
Article Snippet: .. The membranes were blocked with 5% milk in phosphate-buffered saline (PBS) for 1 h, followed by incubation with anti-rabbit VPAC1 primary antibody at a dilution of 1 to 250 or anti-rabbit GAPDH antibody (Sigma-Aldrich, St. Louis, MO) at a ratio of 1 to 10,000 in 1% milk in PBS overnight at 4°C. .. The bound antibodies were detected by horseradish peroxidase-conjugated anti-rabbit Ig secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by ECL detection system (Bio-Rad) per the manufacturer's instructions.

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: .. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody. .. In vivo analysis To express shRNA or Ryk∆PDZ in CST axons, 0.6 μl of shRNA-EGFP-expressing lentivirus or Ryk∆PDZ-EGFP-expressing lentivirus was injected into the motor cortex at 0.8 mm from the midline, and the depth of the injection was 0.5 mm.

Article Title: Repeated Sigma-1 Receptor Antagonist MR309 Administration Modulates Central Neuropathic Pain Development After Spinal Cord Injury in Mice
Article Snippet: Membranes were then incubated with primary antibodies overnight at 4°C: rabbit anti extracellular signal-regulated kinases (total ERK 1/2) (1:40,000, M5670, Sigma-Aldrich) and diphosphorylated ERKs (pERK1/2) (1:800, 44680G, invitrogen) were diluted in T-TBS containing 1% nonfat dry milk. .. To ensure equal protein loading, rabbit anti-GAPDH antibody (1:40,000, Sigma-Aldrich Química S.A.) was used as a loading control.

Article Title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Article Snippet: After 1 h of incubation in blocking buffer (1× PBS and 5% nonfat dry milk), the membranes were probed with affinity-purified rabbit anti-DRA antibody. .. Other primary antibodies used were rat anti-K8 antibody (Troma I, Developmental Studies Hybridoma Bank, University of Iowa), or rabbit-GAPDH antibody (Sigma and Cell Signaling).

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: .. The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ). .. After washing in PBS Tween, the membranes were incubated by HRP-conjugated goat anti-rabbit secondary antibody (1:10,000, Life Technologies) for 1 h at room temperature.

Cell Culture:

Article Title: Kanglaite sensitizes colorectal cancer cells to Taxol via NF-κΒ inhibition and connexin 43 upregulation
Article Snippet: Reagents and antibodies All cell culture media, antibiotics, and trypsin were purchased from Gibco (Grand Island, NY, USA), and foetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). .. Rabbit anti-NF-κΒ p65 antibody, rabbit anti-IKKα antibody, rabbit anti-IκΒα antibody, rabbit anti-caspase-8 antibody, rabbit anti-GAPDH antibody, rabbit anti-Poly[ADP-ribose] Polymerase (PARP) antibody, rabbit anti-cyclin B1 antibody, rabbit anti-survivin antibody, rabbit anti-caspase-3 antibody, mouse anti-α-tubulin antibody, rabbit anti-β-actin antibody, goat anti-rabbit IgG-peroxidase, goat anti-mouse IgG-peroxidase, goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC, DAPI, methyl thiazolyl tetrazolium (MTT), and Taxol were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Lysis:

Article Title: Kanglaite sensitizes colorectal cancer cells to Taxol via NF-κΒ inhibition and connexin 43 upregulation
Article Snippet: Rabbit anti-NF-κΒ p65 antibody, rabbit anti-IKKα antibody, rabbit anti-IκΒα antibody, rabbit anti-caspase-8 antibody, rabbit anti-GAPDH antibody, rabbit anti-Poly[ADP-ribose] Polymerase (PARP) antibody, rabbit anti-cyclin B1 antibody, rabbit anti-survivin antibody, rabbit anti-caspase-3 antibody, mouse anti-α-tubulin antibody, rabbit anti-β-actin antibody, goat anti-rabbit IgG-peroxidase, goat anti-mouse IgG-peroxidase, goat anti-rabbit IgG-FITC, goat anti-mouse IgG-FITC, DAPI, methyl thiazolyl tetrazolium (MTT), and Taxol were purchased from Sigma-Aldrich (St. Louis, MO, USA). .. ECL Plus substrate, bicinchoninic acid (BCA) reagents, and RIPA lysis buffer were purchased from CWBio (Beijing, China).

Sequencing:

Article Title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Article Snippet: The affinity-purified DRA antibody was raised against the COOH-terminal amino acid (745–764) sequence INTNGGLRNRVYEPVETKF of SLC26A3 (accession number: ) at Research Resource Center, University of Illinois at Chicago ( ). .. Other primary antibodies used were rat anti-K8 antibody (Troma I, Developmental Studies Hybridoma Bank, University of Iowa), or rabbit-GAPDH antibody (Sigma and Cell Signaling).

Sonication:

Article Title: Repeated Sigma-1 Receptor Antagonist MR309 Administration Modulates Central Neuropathic Pain Development After Spinal Cord Injury in Mice
Article Snippet: Spinal cord tissue was homogenized by sonication in TRIS buffer (50 mM Tris, 150 nM NaCl, 1% NP-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, Triton X-100, 0.1% SDS, 1 mM Na3 VO4 , 25mM Na), 5% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail, all from Sigma-Aldrich Química S.A. .. To ensure equal protein loading, rabbit anti-GAPDH antibody (1:40,000, Sigma-Aldrich Química S.A.) was used as a loading control.

Affinity Purification:

Article Title: Keratin 8 knockdown leads to loss of the chloride transporter DRA in the colon
Article Snippet: The affinity-purified DRA antibody was raised against the COOH-terminal amino acid (745–764) sequence INTNGGLRNRVYEPVETKF of SLC26A3 (accession number: ) at Research Resource Center, University of Illinois at Chicago ( ). .. Other primary antibodies used were rat anti-K8 antibody (Troma I, Developmental Studies Hybridoma Bank, University of Iowa), or rabbit-GAPDH antibody (Sigma and Cell Signaling).

Protease Inhibitor:

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation
Article Snippet: Western blotting Cells were washed twice with PBS and lysed in RIPA buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% SDS, and 1 mM EDTA), containing 1 protease inhibitor cocktail tablet (Roche) per 10 ml of buffer. .. Equal loading was confirmed by probing the blots with antibodies against GAPDH (rabbit polyclonal, 1 : 5000) or β -actin (mouse monoclonal, 1 : 10 000), both from Sigma, Dorset, UK.

Article Title: Repeated Sigma-1 Receptor Antagonist MR309 Administration Modulates Central Neuropathic Pain Development After Spinal Cord Injury in Mice
Article Snippet: Spinal cord tissue was homogenized by sonication in TRIS buffer (50 mM Tris, 150 nM NaCl, 1% NP-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, Triton X-100, 0.1% SDS, 1 mM Na3 VO4 , 25mM Na), 5% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail, all from Sigma-Aldrich Química S.A. .. To ensure equal protein loading, rabbit anti-GAPDH antibody (1:40,000, Sigma-Aldrich Química S.A.) was used as a loading control.

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: Pontine slices (300 μm thick) containing the LC area were obtained from 3- to 4-week-old mice with a vibratome sectioning system, and the pons was processed in radioimmune precipitation assay buffer (Sigma-Aldrich) with 1% protease inhibitor. .. The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ).

Immunofluorescence:

Article Title: Distinct Functions for Mammalian CLASP1 and -2 During Neurite and Axon Elongation
Article Snippet: Antibodies We used the following commercial primary antibodies (Abs): mouse anti-α-tubulin, mouse anti-β-tubulin, mouse anti-α-Tyrosinated-tubulin, mouse anti-α-Acetylated tubulin, mouse anti-β-actin, and rabbit anti-GAPDH (all from Sigma-Aldrich); mouse anti-βIII tubulin (Promega), mouse anti-α-detyrosinated (Glu)-tubulin (Abcam); mouse anti-GFP (Roche); rabbit anti-Akt-phosphoSer (Ser 473), rabbit anti-GSK3-α/β-phosphoSer (Ser 21/Ser9), rabbit anti-GSK3-α/β (Cell Signaling), rat anti-CLASP1 (#1A6) and -CLASP2 (#12H2, Absea antibodies). .. We also used previously described primary rabbit anti-CLASP1 [#402, for immunofluorescence (IF), and #2292 for western blot (WB)], and rabbit anti-CLASP2 (#2358, both for IF and WB; Akhmanova et al., ).

SDS Page:

Article Title: Ryk regulates Wnt5a repulsion of mouse corticospinal tract through modulating planar cell polarity signaling
Article Snippet: The protein samples were separated by 10% SDS-PAGE and transferred onto PVDF membranes. .. After blocking in blocking buffer (0.5% milk) for 1 h at room temperature, the blots were incubated with rabbit anti-Ryk (1:500), goat anti-Vangl2 (1:500), rabbit anti-Fzd3 (1:500), mouse anti-Dvl1 (1:200), rabbit anti-GAPDH (1:1000; Sigma-Aldrich), mouse anti-E-cadherin (1:1000), mouse anti-FLAG (1:1000) or rabbit anti-HA (1:1000), and subsequently with a peroxidase-conjugated secondary antibody.

Article Title: Sulphoxythiocarbamates modify cysteine residues in HSP90 causing degradation of client proteins and inhibition of cancer cell proliferation
Article Snippet: Proteins were resolved by SDS–PAGE, transferred to immobilon-P membranes, and probed with specific antibodies against HSP70 (mouse monoclonal, 1 : 1000, StressMarq, York, UK), HSP90 (mouse monoclonal, 1 : 5000, BD Biosciences, Franklin Lakes, NJ, USA), HER2 (rabbit polyclonal, 1 : 500, Millipore, Temecula, CA, USA), RAF1 (rabbit polyclonal, 1 : 200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), GSK3 (mouse monoclonal, 1 : 1000, Biosource, Camarillo, CA, USA), CHK1 (mouse monoclonal, 1 : 1000, Santa Cruz Biotechnology, Camarillo, CA, USA), pS345-CHK1 (rabbit polyclonal, 1 : 1000, Cell Signaling, Danvers, MA, USA), CDK1 (rabbit polyclonal, 1 : 1000, Cell Signaling), or p53 (DO-1) (mouse monoclonal, 1 : 1000, Abcam, Cambridge, UK). .. Equal loading was confirmed by probing the blots with antibodies against GAPDH (rabbit polyclonal, 1 : 5000) or β -actin (mouse monoclonal, 1 : 10 000), both from Sigma, Dorset, UK.

Article Title: Repeated Sigma-1 Receptor Antagonist MR309 Administration Modulates Central Neuropathic Pain Development After Spinal Cord Injury in Mice
Article Snippet: Thirty micrograms were fractionated by 10% (w/v) SDS-PAGE and transferred onto a polyvinylidene difluoride membrane, then blocked, with either 5% nonfat dry milk or bovine serum albumin (BSA), in Tris-Tween 20-buffered saline (T-TBS) for 1 h at room temperature. .. To ensure equal protein loading, rabbit anti-GAPDH antibody (1:40,000, Sigma-Aldrich Química S.A.) was used as a loading control.

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: BCA protein assay reagent (Pierce) was used to estimate the protein concentrations, and 30 μg of proteins was used to detect δ subunit signals in 10% SDS-PAGE gels and electrophoretically transferred to nitrocellulose membranes. .. The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ).

Software:

Article Title: Subchronic Administration and Combination Metabotropic Glutamate and GABAB Receptor Drug Therapy in Fragile X Syndrome
Article Snippet: The following antibodies were used: mouse anti-GABAB R1 (clone NR3A/49; 1:250; NeuroMab, University of California, Davis, CA/National Institutes of Health, Bethesda, MD); mouse anti-GABAB R2 (clone N81/2; 1:750; NeuroMab, University of California, Davis/National Institutes of Health); rabbit anti-mGluR5 (1:500; Millipore Bioscience Research Reagents, Temecula, CA); mouse anti-GAPDH antibody (1:40,000–1:100,000; Sigma-Aldrich); and a goat anti-mouse (The Jackson Laboratory, Bar Harbor, ME) or goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA) horseradish peroxidase-conjugated secondary antibody. .. Densitometric analysis was carried out using AlphaEaseFC software (Alpha Innotech).

Article Title: Methyl CpG Binding Protein 2 Gene Disruption Augments Tonic Currents of γ-Aminobutyric Acid Receptors in Locus Coeruleus Neurons
Article Snippet: The membranes were then blocked for 2 h in 5% nonfat milk and incubated overnight at 4 °C with rabbit GAPDH primary antibody (1:10,000, Sigma-Aldrich) and rabbit δ subunit primary antibody (1:1000, EMD Millipore, Billerica, MA) ( ). .. The immunoblotting signals were quantified using ImageJ software (National Institutes of Health).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    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: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lc3b ii gapdh/product/Millipore
    Average 93 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    lc3b ii gapdh - by Bioz Stars, 2020-04
    93/100 stars
      Buy from Supplier

    99
    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
    https://www.bioz.com/result/anti gapdh/product/Millipore
    Average 99 stars, based on 641 article reviews
    Price from $9.99 to $1999.99
    anti gapdh - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    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