Structured Review

Cell Signaling Technology Inc glyceraldehyde 3 phosphate dehydrogenase gapdh
Expression of peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) is over-expressed in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A) Immunofluorescence staining of PGC-1β in primary cultures of FLS from osteoarthritis (OA) and RA patients. (a, DAPI (blue); b, PGC-1β (green); c, neutral light; d, merged a, b with c. a, b, c: original magnification × 400). (B) Left panel: PGC-1β mRNA expression in FLS from RA (n = 8) compared with that from OA (n =6) evaluated by qPCR. Right panel: PGC-1β protein level in FLS from RA patients (n = 8) and OA controls (n = 6) was detected by western blot. The intensity for each band was densitometrically quantified and normalized against the intensity of <t>glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH).</t> The data are represented by mean ± SD. * P
Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/glyceraldehyde 3 phosphate dehydrogenase gapdh/product/Cell Signaling Technology Inc
Average 99 stars, based on 5 article reviews
Price from $9.99 to $1999.99
glyceraldehyde 3 phosphate dehydrogenase gapdh - by Bioz Stars, 2020-02
99/100 stars

Images

1) Product Images from "Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation"

Article Title: Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-014-0472-6

Expression of peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) is over-expressed in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A) Immunofluorescence staining of PGC-1β in primary cultures of FLS from osteoarthritis (OA) and RA patients. (a, DAPI (blue); b, PGC-1β (green); c, neutral light; d, merged a, b with c. a, b, c: original magnification × 400). (B) Left panel: PGC-1β mRNA expression in FLS from RA (n = 8) compared with that from OA (n =6) evaluated by qPCR. Right panel: PGC-1β protein level in FLS from RA patients (n = 8) and OA controls (n = 6) was detected by western blot. The intensity for each band was densitometrically quantified and normalized against the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data are represented by mean ± SD. * P
Figure Legend Snippet: Expression of peroxisome proliferator-activated receptor-gamma coactivator-1 β (PGC-1β) is over-expressed in rheumatoid arthritis (RA)-fibrolast-like synoviocytes (FLS). (A) Immunofluorescence staining of PGC-1β in primary cultures of FLS from osteoarthritis (OA) and RA patients. (a, DAPI (blue); b, PGC-1β (green); c, neutral light; d, merged a, b with c. a, b, c: original magnification × 400). (B) Left panel: PGC-1β mRNA expression in FLS from RA (n = 8) compared with that from OA (n =6) evaluated by qPCR. Right panel: PGC-1β protein level in FLS from RA patients (n = 8) and OA controls (n = 6) was detected by western blot. The intensity for each band was densitometrically quantified and normalized against the intensity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The data are represented by mean ± SD. * P

Techniques Used: Expressing, Pyrolysis Gas Chromatography, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Western Blot

2) Product Images from "Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition"

Article Title: Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition

Journal: The British Journal of Ophthalmology

doi: 10.1136/bjophthalmol-2018-311937

Expression of secreted protein acidic cysteine (SPARC) and SPARC-regulated proteins in the mouse model of conjunctival scarring treated with small interfering RNA. (A) Immunoblot analyses of SPARC, collagen 1A1 (COL1A1), fibronectin 1 (FN1) and matrix metalloproteinase (MMP)-14 expression on treatment with either siScram or siSparc for 7 days. (B) Immunoblot analyses of SPARC, COL1A1, FN1 and MMP-14 expression on treatment with either siScram or siSparc for 14 days. Densitometry value of each protein band, expressed as ratio with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the same immunoblot, is shown below the immunoblots. The mean fold change±SD of the three densitometric ratios for each condition is shown. Fold change comparing the two treatments calculated from the means of the two groups is also shown. *P
Figure Legend Snippet: Expression of secreted protein acidic cysteine (SPARC) and SPARC-regulated proteins in the mouse model of conjunctival scarring treated with small interfering RNA. (A) Immunoblot analyses of SPARC, collagen 1A1 (COL1A1), fibronectin 1 (FN1) and matrix metalloproteinase (MMP)-14 expression on treatment with either siScram or siSparc for 7 days. (B) Immunoblot analyses of SPARC, COL1A1, FN1 and MMP-14 expression on treatment with either siScram or siSparc for 14 days. Densitometry value of each protein band, expressed as ratio with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) from the same immunoblot, is shown below the immunoblots. The mean fold change±SD of the three densitometric ratios for each condition is shown. Fold change comparing the two treatments calculated from the means of the two groups is also shown. *P

Techniques Used: Expressing, Small Interfering RNA, Western Blot

3) Product Images from "Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells"

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19051498

Effect of transforming growth factor β (TGFβ 2 ) on SK-1, phospho-Smad2, connective tissue growth factor (CTGF), and fibronectin expression in HK2 cells. ( A ) HK2 cells were taken unstimulated (−) or stimulated with 5 ng/mL of TGFβ 2 for the indicated time periods up to 24 h. Thereafter, cell lysates were prepared and equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and subjected to Western blotting using antibodies against SK-1 (1:2000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000), phospho-Smad2 (1:1000), CTGF (1:500), and fibronectin (1:500). To detect secreted CTGF, equal volumes of supernatants of stimulated cells were taken for protein precipitation by trichloroacetic acid (TCA) and were then analysed the same way as described for lysates using the CTGF antibody. Band intensities were evaluated by a LICOR R imaging system. Data in ( B – F ) show the evaluated respective bands. Results are expressed as fold increase and are means ± S.D. ( n = 3); * p
Figure Legend Snippet: Effect of transforming growth factor β (TGFβ 2 ) on SK-1, phospho-Smad2, connective tissue growth factor (CTGF), and fibronectin expression in HK2 cells. ( A ) HK2 cells were taken unstimulated (−) or stimulated with 5 ng/mL of TGFβ 2 for the indicated time periods up to 24 h. Thereafter, cell lysates were prepared and equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and subjected to Western blotting using antibodies against SK-1 (1:2000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000), phospho-Smad2 (1:1000), CTGF (1:500), and fibronectin (1:500). To detect secreted CTGF, equal volumes of supernatants of stimulated cells were taken for protein precipitation by trichloroacetic acid (TCA) and were then analysed the same way as described for lysates using the CTGF antibody. Band intensities were evaluated by a LICOR R imaging system. Data in ( B – F ) show the evaluated respective bands. Results are expressed as fold increase and are means ± S.D. ( n = 3); * p

Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Imaging

4) Product Images from "Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice"

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113399

Quantification of proteins in the myocardium by western blot. Bar graphs illustrating ACC ( A ), p-ACC ( B ), TGF-β1 ( C ), and osteopontin ( D ) protein levels quantified by western blot in the myocardium. SC diet mice and CC diet mice are indicated by open bars and closed bars, respectively. Representative images of western blots are shown in panel E. All protein levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. All data represent means ± SEM ( n = 6).
Figure Legend Snippet: Quantification of proteins in the myocardium by western blot. Bar graphs illustrating ACC ( A ), p-ACC ( B ), TGF-β1 ( C ), and osteopontin ( D ) protein levels quantified by western blot in the myocardium. SC diet mice and CC diet mice are indicated by open bars and closed bars, respectively. Representative images of western blots are shown in panel E. All protein levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. All data represent means ± SEM ( n = 6).

Techniques Used: Western Blot, Mouse Assay

5) Product Images from "Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy"

Article Title: Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy

Journal: Science translational medicine

doi: 10.1126/scitranslmed.3003293

Effect of fasting on genes involved in growth and proliferation. ( A and B ) Microarray analysis on the liver, heart, skeletal muscle, and subcutaneous breast tumors (4T1) from normally fed or fasted (48 hours) mice of cellular proliferation pathways (A) and translational machinery including ribosome assembly/biogenesis (B). ( C ) Effect of starvation on protein concentration in 4T1, B16, and GL26 cells ( n = 3). ( D ) Effect of starvation on the rate of AHA (methionine analog) incorporation in 4T1 cells ( n = 3). Data are means ± SEM [(C) and (D)]. ( E and F ) Effect of fasting on Akt, S6K, and eIF2α phosphorylation in murine breast cancer cells (4T1) in vivo (E) and in vitro (F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P
Figure Legend Snippet: Effect of fasting on genes involved in growth and proliferation. ( A and B ) Microarray analysis on the liver, heart, skeletal muscle, and subcutaneous breast tumors (4T1) from normally fed or fasted (48 hours) mice of cellular proliferation pathways (A) and translational machinery including ribosome assembly/biogenesis (B). ( C ) Effect of starvation on protein concentration in 4T1, B16, and GL26 cells ( n = 3). ( D ) Effect of starvation on the rate of AHA (methionine analog) incorporation in 4T1 cells ( n = 3). Data are means ± SEM [(C) and (D)]. ( E and F ) Effect of fasting on Akt, S6K, and eIF2α phosphorylation in murine breast cancer cells (4T1) in vivo (E) and in vitro (F). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P

Techniques Used: Microarray, Mouse Assay, Protein Concentration, In Vivo, In Vitro

6) Product Images from "Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces"

Article Title: Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20246221

Expression of yes-associated protein (YAP) and phospho-YAP (pYAP) appeared to coincide with Dsg3 in response to cyclic strain. ( A ) Western blotting for HaCaT cells with strained, or non-strained, for 6 h. Lysates were extracted either immediately (0 h) after the strain or later, after being transferred to a stationary state, at various time points as indicated. The timeline for this experiment is shown above the blots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) Band densitometry analysis for Dsg3, pYAP, and YAP expression. Note that all three proteins showed increased expression in response to cyclic strain compared to non-strained samples. Quantitation for each protein was normalized against the loading in each lane and presented as a fold change of the non-strained sample at the 0 h time point ( n = 3, mean ± S.E.M, * p
Figure Legend Snippet: Expression of yes-associated protein (YAP) and phospho-YAP (pYAP) appeared to coincide with Dsg3 in response to cyclic strain. ( A ) Western blotting for HaCaT cells with strained, or non-strained, for 6 h. Lysates were extracted either immediately (0 h) after the strain or later, after being transferred to a stationary state, at various time points as indicated. The timeline for this experiment is shown above the blots. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. ( B ) Band densitometry analysis for Dsg3, pYAP, and YAP expression. Note that all three proteins showed increased expression in response to cyclic strain compared to non-strained samples. Quantitation for each protein was normalized against the loading in each lane and presented as a fold change of the non-strained sample at the 0 h time point ( n = 3, mean ± S.E.M, * p

Techniques Used: Expressing, Western Blot, Quantitation Assay

7) Product Images from "Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells"

Article Title: Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells

Journal: Journal of Breast Cancer

doi: 10.4048/jbc.2015.18.2.112

Isocryptotanshinone (ICTS) induced apoptosis in MCF-7 cells. (A) Cells were treated with indicated concentrations of ICTS for 24 hours, and the nuclei were stained by Hoechst 33342. Arrows indicated the condensed nuclei in cells. (B) DNA fragmentation assay was performed in MCF-7 cells after treatment with indicated concentrations of ICTS for 24 hours. (C, D) MCF-7 cells were treated with ICTS (0-20 µM) for 24 hours, and the expressions of Bcl-2, Bcl-XL, BAK, and BAX, as well as poly-ADP-ribose polymerase (PARP), caspase-3, and caspase-9 were detected by Western blot. GAPDH=glyceraldehyde 3-phosphate dehydrogenase.
Figure Legend Snippet: Isocryptotanshinone (ICTS) induced apoptosis in MCF-7 cells. (A) Cells were treated with indicated concentrations of ICTS for 24 hours, and the nuclei were stained by Hoechst 33342. Arrows indicated the condensed nuclei in cells. (B) DNA fragmentation assay was performed in MCF-7 cells after treatment with indicated concentrations of ICTS for 24 hours. (C, D) MCF-7 cells were treated with ICTS (0-20 µM) for 24 hours, and the expressions of Bcl-2, Bcl-XL, BAK, and BAX, as well as poly-ADP-ribose polymerase (PARP), caspase-3, and caspase-9 were detected by Western blot. GAPDH=glyceraldehyde 3-phosphate dehydrogenase.

Techniques Used: Staining, DNA Fragmentation Assay, Western Blot

8) Product Images from "Anorexic response to rapamycin does not appear to involve a central mechanism"

Article Title: Anorexic response to rapamycin does not appear to involve a central mechanism

Journal: Clinical and experimental pharmacology & physiology

doi: 10.1111/1440-1681.12601

Phosphorylated S6 (pS6), total S6 (tS6), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein derived from Western blots in brain and liver of intracerebroventricular (icv) vehicle (control; open bars) or rapamycin (solid bars) infused old rats. N=6 rats/group
Figure Legend Snippet: Phosphorylated S6 (pS6), total S6 (tS6), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein derived from Western blots in brain and liver of intracerebroventricular (icv) vehicle (control; open bars) or rapamycin (solid bars) infused old rats. N=6 rats/group

Techniques Used: Derivative Assay, Western Blot

9) Product Images from "The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats"

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats

Journal: PLoS ONE

doi: 10.1371/journal.pone.0057757

Effects of CGEN-856S and Ang-(1–7) administration on AKT phosphorylation and on the quantity of p-AKT. (A) Representative immunoblots demonstrating the presence of Mas in Mas-transfected CHO cells (CHO-Mas) and the absence of Mas in untransfected cells (CHO-K1). (B) Effects of CGEN-856S and Ang-(1–7) administration (10 −9 and 10 −7 mol/L) for 10 min on p-AKT levels in CHO-Mas cells. (C) The absence of effects of CGEN-856S and Ang-(1–7) administration (10 −7 mol/L) for 10 min on p-AKT levels in CHO-K1 cells. (D) Effects of CGEN-856S and Ang-(1–7) administration (10 −9 mol/L) for 5 min on AKT phosphorylation in CHO-Mas cells. Ang-(1–7) (10 −9 and 10 −7 mol/L) was used as a positive control and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and total AKT were used as loading controls. * P
Figure Legend Snippet: Effects of CGEN-856S and Ang-(1–7) administration on AKT phosphorylation and on the quantity of p-AKT. (A) Representative immunoblots demonstrating the presence of Mas in Mas-transfected CHO cells (CHO-Mas) and the absence of Mas in untransfected cells (CHO-K1). (B) Effects of CGEN-856S and Ang-(1–7) administration (10 −9 and 10 −7 mol/L) for 10 min on p-AKT levels in CHO-Mas cells. (C) The absence of effects of CGEN-856S and Ang-(1–7) administration (10 −7 mol/L) for 10 min on p-AKT levels in CHO-K1 cells. (D) Effects of CGEN-856S and Ang-(1–7) administration (10 −9 mol/L) for 5 min on AKT phosphorylation in CHO-Mas cells. Ang-(1–7) (10 −9 and 10 −7 mol/L) was used as a positive control and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and total AKT were used as loading controls. * P

Techniques Used: Western Blot, Transfection, Positive Control

10) Product Images from "Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice"

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113399

Quantification of proteins in the myocardium by western blot. Bar graphs illustrating ACC ( A ), p-ACC ( B ), TGF-β1 ( C ), and osteopontin ( D ) protein levels quantified by western blot in the myocardium. SC diet mice and CC diet mice are indicated by open bars and closed bars, respectively. Representative images of western blots are shown in panel E. All protein levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. All data represent means ± SEM ( n = 6).
Figure Legend Snippet: Quantification of proteins in the myocardium by western blot. Bar graphs illustrating ACC ( A ), p-ACC ( B ), TGF-β1 ( C ), and osteopontin ( D ) protein levels quantified by western blot in the myocardium. SC diet mice and CC diet mice are indicated by open bars and closed bars, respectively. Representative images of western blots are shown in panel E. All protein levels were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein level. All data represent means ± SEM ( n = 6).

Techniques Used: Western Blot, Mouse Assay

11) Product Images from "Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages"

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages

Journal: Mediators of Inflammation

doi: 10.1155/2017/9290416

Effects of regular voluntary exercise (VE) on the protein levels of prointerleukin- (IL-) 1 β and pro-IL-18 in macrophages stimulated with or without lipopolysaccharide (LPS). Peritoneal-exudate macrophages isolated from the sedentary control (SC) and VE mice were cultured for 6 h in the presence or absence of 100 ng/mL LPS. (a) The protein levels of pro-IL-1 β and pro-IL-18 were analyzed by western blotting. (b) The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Mean ± standard error of the mean (SEM; n = 3-4) values are provided. ∗ p
Figure Legend Snippet: Effects of regular voluntary exercise (VE) on the protein levels of prointerleukin- (IL-) 1 β and pro-IL-18 in macrophages stimulated with or without lipopolysaccharide (LPS). Peritoneal-exudate macrophages isolated from the sedentary control (SC) and VE mice were cultured for 6 h in the presence or absence of 100 ng/mL LPS. (a) The protein levels of pro-IL-1 β and pro-IL-18 were analyzed by western blotting. (b) The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Mean ± standard error of the mean (SEM; n = 3-4) values are provided. ∗ p

Techniques Used: Isolation, Mouse Assay, Cell Culture, Western Blot

Effects of regular voluntary exercise (VE) on the protein levels of procaspase-8 in macrophages. Immediately after peritoneal-exudate macrophages were isolated from the sedentary control (SC) and VE mice, whole cellular proteins were extracted. The protein levels of procaspase-8 were analyzed by western blotting (upper panel). The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lower panel). Mean ± standard error of the mean (SEM; n = 4) values are provided.
Figure Legend Snippet: Effects of regular voluntary exercise (VE) on the protein levels of procaspase-8 in macrophages. Immediately after peritoneal-exudate macrophages were isolated from the sedentary control (SC) and VE mice, whole cellular proteins were extracted. The protein levels of procaspase-8 were analyzed by western blotting (upper panel). The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lower panel). Mean ± standard error of the mean (SEM; n = 4) values are provided.

Techniques Used: Isolation, Mouse Assay, Western Blot

Effects of regular voluntary exercise (VE) on the expression levels of inflammasome components in macrophages. Immediately after peritoneal-exudate macrophages were isolated from the sedentary control (SC) and VE mice, whole cellular proteins and total cellular RNA were extracted. (a) The protein levels of Nod-like receptor family pyrin domain containing 3 (NLRP3) were analyzed by western blotting after immunoprecipitation (IP) (upper panel). The protein levels of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (b) and procaspase-1 (c) were analyzed by western blotting (upper panel). The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lower panel). The mRNA levels of caspase-1 (d) and NLRP3 (e) were analyzed by real-time polymerase chain reaction (PCR). The mRNA levels are presented as ratios relative to the levels of 18S rRNA. Mean ± standard error of the mean (SEM; n = 4) values are provided. ∗∗ p
Figure Legend Snippet: Effects of regular voluntary exercise (VE) on the expression levels of inflammasome components in macrophages. Immediately after peritoneal-exudate macrophages were isolated from the sedentary control (SC) and VE mice, whole cellular proteins and total cellular RNA were extracted. (a) The protein levels of Nod-like receptor family pyrin domain containing 3 (NLRP3) were analyzed by western blotting after immunoprecipitation (IP) (upper panel). The protein levels of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) (b) and procaspase-1 (c) were analyzed by western blotting (upper panel). The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lower panel). The mRNA levels of caspase-1 (d) and NLRP3 (e) were analyzed by real-time polymerase chain reaction (PCR). The mRNA levels are presented as ratios relative to the levels of 18S rRNA. Mean ± standard error of the mean (SEM; n = 4) values are provided. ∗∗ p

Techniques Used: Expressing, Isolation, Mouse Assay, Western Blot, Immunoprecipitation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Effects of regular voluntary exercise (VE) on the activation of intracellular signaling downstream of TLR4 in macrophages stimulated with or without lipopolysaccharide (LPS). Peritoneal-exudate macrophages isolated from the sedentary control (SC) and VE mice were cultured for 20 min in the presence or absence of 100 ng/mL LPS. The protein levels of inhibitor of κ B (I κ B) α (a) and phosphorylation levels of I κ B kinase (IKK) β (b), p54 (c), p46 (d), and p38 (e) were analyzed by western blotting (upper panel). The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lower panel), whereas the phosphorylation levels are presented as ratios relative to the respective protein levels (lower panel). Mean ± standard error of the mean (SEM; n = 4) values are provided. ∗∗ p
Figure Legend Snippet: Effects of regular voluntary exercise (VE) on the activation of intracellular signaling downstream of TLR4 in macrophages stimulated with or without lipopolysaccharide (LPS). Peritoneal-exudate macrophages isolated from the sedentary control (SC) and VE mice were cultured for 20 min in the presence or absence of 100 ng/mL LPS. The protein levels of inhibitor of κ B (I κ B) α (a) and phosphorylation levels of I κ B kinase (IKK) β (b), p54 (c), p46 (d), and p38 (e) were analyzed by western blotting (upper panel). The protein levels are presented as ratios relative to the levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (lower panel), whereas the phosphorylation levels are presented as ratios relative to the respective protein levels (lower panel). Mean ± standard error of the mean (SEM; n = 4) values are provided. ∗∗ p

Techniques Used: Activation Assay, Isolation, Mouse Assay, Cell Culture, Western Blot

12) Product Images from "Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B"

Article Title: Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B

Journal: Molecular Therapy

doi: 10.1016/j.ymthe.2017.06.025

Western Blot Analysis of Dysferlin and MG53 Protein Expression in Striated Muscle Tissue from Wild-Type and Dysf −/− Mice (A and C) Western blots of dysferlin and MG53 levels in whole gluteus (A) and gastrocnemius (C) tissue extracts, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control, from C57BL/6J (WT, lane 1) and Dysf −/− mice (sedentary group, lanes 2–4; exercised saline-treated group, lanes 5–7; exercised rhMG53-treated group, lanes 8–10). (B and D) Densitometric analysis of the western blots presented in (A) and (C) was carried out using ImageJ software. The intensity of MG53 bands was normalized to the GAPDH band intensity of the respective sample. Data are means ± SEM (n = 3 for each group).
Figure Legend Snippet: Western Blot Analysis of Dysferlin and MG53 Protein Expression in Striated Muscle Tissue from Wild-Type and Dysf −/− Mice (A and C) Western blots of dysferlin and MG53 levels in whole gluteus (A) and gastrocnemius (C) tissue extracts, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a control, from C57BL/6J (WT, lane 1) and Dysf −/− mice (sedentary group, lanes 2–4; exercised saline-treated group, lanes 5–7; exercised rhMG53-treated group, lanes 8–10). (B and D) Densitometric analysis of the western blots presented in (A) and (C) was carried out using ImageJ software. The intensity of MG53 bands was normalized to the GAPDH band intensity of the respective sample. Data are means ± SEM (n = 3 for each group).

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

13) Product Images from "Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells"

Article Title: Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells

Journal: Oncology Reports

doi: 10.3892/or.2015.3990

Contribution of endogenous Lck kinase activity in its nuclear translocation. (A) Nuclear and cytosolic fractions were isolated from Jurkat and Jcam cells either unstimulated (Un) or stimulated with pervanadate for 5 min (S). Equal amount of proteins from each set of lysates was immunoprecipitated (IP) with an anti-Lck antibody, and then subjected to immunoblotting with an anti-phospho-Src family (Y416) antibody to visualize Lck phosphorylation (pLck). The amount of total Lck was determined by sequential blotting with the anti-Lck antibody. A fraction of the total lysates was probed for GAPDH and lamin B1 to verify fraction purity. (B) Jurkat cells were treated with dasatinib or vehicle control for 2 h. Whole cell lysates (WCL) were prepared from a fraction of the cells and analyzed by immunoblotting using antibodies specific for phospho-Y416-Src family (pSrc) and GAPDH (lanes 1 and 2). Nuclear proteins isolated from the remaining cells were subjected to immunoblotting using antibodies specific for Lck, lamin B1 and Eps15 (cytosolic marker) (lanes 5 and 6). Equal amount of whole cell lysates was also analyzed by immunoblotting using antibodies specific for Lck, lamin B1 and Eps15 (lanes 3 and 4). Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Eps15, epidermal growth factor receptor substrate 15.
Figure Legend Snippet: Contribution of endogenous Lck kinase activity in its nuclear translocation. (A) Nuclear and cytosolic fractions were isolated from Jurkat and Jcam cells either unstimulated (Un) or stimulated with pervanadate for 5 min (S). Equal amount of proteins from each set of lysates was immunoprecipitated (IP) with an anti-Lck antibody, and then subjected to immunoblotting with an anti-phospho-Src family (Y416) antibody to visualize Lck phosphorylation (pLck). The amount of total Lck was determined by sequential blotting with the anti-Lck antibody. A fraction of the total lysates was probed for GAPDH and lamin B1 to verify fraction purity. (B) Jurkat cells were treated with dasatinib or vehicle control for 2 h. Whole cell lysates (WCL) were prepared from a fraction of the cells and analyzed by immunoblotting using antibodies specific for phospho-Y416-Src family (pSrc) and GAPDH (lanes 1 and 2). Nuclear proteins isolated from the remaining cells were subjected to immunoblotting using antibodies specific for Lck, lamin B1 and Eps15 (cytosolic marker) (lanes 5 and 6). Equal amount of whole cell lysates was also analyzed by immunoblotting using antibodies specific for Lck, lamin B1 and Eps15 (lanes 3 and 4). Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Eps15, epidermal growth factor receptor substrate 15.

Techniques Used: Activity Assay, Translocation Assay, Isolation, Immunoprecipitation, Marker

Nuclear localization of endogenous Lck in T cell lines and primary cells. (A) Nuclear (Nuc) fractions isolated from Jurkat and LSTRA cell lines were analyzed by Lck immunoblotting. GAPDH (cytosolic marker) and lamin B1 (nuclear marker) immunoblotting were also performed to verify the purity of the nuclear fraction. Jurkat whole cell lysate (WCL) was used as a positive control for markers (lane 3). (B) Jurkat cells were subjected to immunofluorescence microscopy using an anti-Lck antibody (red). Nuclei were counterstained with DAPI (blue). Nuclear staining of Lck is indicated by an arrow in the enlarged merged image. Scale bars of 5 μ m are shown at the bottom of the microscopy images. (C) Mouse splenocytes stimulated with pervanadate for 5 min (+PV) or left unstimulated (−PV) were analyzed by anti-Lck immunofluorescence microscopy (green) with DAPI counterstain (blue). Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure Legend Snippet: Nuclear localization of endogenous Lck in T cell lines and primary cells. (A) Nuclear (Nuc) fractions isolated from Jurkat and LSTRA cell lines were analyzed by Lck immunoblotting. GAPDH (cytosolic marker) and lamin B1 (nuclear marker) immunoblotting were also performed to verify the purity of the nuclear fraction. Jurkat whole cell lysate (WCL) was used as a positive control for markers (lane 3). (B) Jurkat cells were subjected to immunofluorescence microscopy using an anti-Lck antibody (red). Nuclei were counterstained with DAPI (blue). Nuclear staining of Lck is indicated by an arrow in the enlarged merged image. Scale bars of 5 μ m are shown at the bottom of the microscopy images. (C) Mouse splenocytes stimulated with pervanadate for 5 min (+PV) or left unstimulated (−PV) were analyzed by anti-Lck immunofluorescence microscopy (green) with DAPI counterstain (blue). Lck, lymphocyte-specific protein tyrosine kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Techniques Used: Isolation, Marker, Positive Control, Immunofluorescence, Microscopy, Staining

14) Product Images from "IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p"

Article Title: IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-018-0959-1

Differential expression of selected microarray-identified long non-coding RNAs (lncRNAs) and their binding to insulin-like growth factor 2 messenger RNA binding protein (IMP1). a Total RNA was extracted from MDA231 cells expressing green fluorescent protein (GFP) or Flag-tagged IMP1-GFP. RT-qPCR was used to analyze the levels of six microarray-identified lncRNAs. Relative levels of the lncRNAs were nomalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed. The data are presented as means ± SD from three independent experiments: * P
Figure Legend Snippet: Differential expression of selected microarray-identified long non-coding RNAs (lncRNAs) and their binding to insulin-like growth factor 2 messenger RNA binding protein (IMP1). a Total RNA was extracted from MDA231 cells expressing green fluorescent protein (GFP) or Flag-tagged IMP1-GFP. RT-qPCR was used to analyze the levels of six microarray-identified lncRNAs. Relative levels of the lncRNAs were nomalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed. The data are presented as means ± SD from three independent experiments: * P

Techniques Used: Expressing, Microarray, Binding Assay, RNA Binding Assay, Quantitative RT-PCR

Interaction of insulin-like growth factor 2 messenger RNA binding protein (IMP1) with urethral carcinoma-associated 1 (UCA1) in breast cancer cells. a Upper: representitive western blots indicate the precipitation (IP) of IMP1 in T47D cells from co-IP experiments; lower: RT-qPCR shows that UCA1 was preferentially co-precipitated with IMP1 antibody relative to the IgG control. Data are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed: ** P
Figure Legend Snippet: Interaction of insulin-like growth factor 2 messenger RNA binding protein (IMP1) with urethral carcinoma-associated 1 (UCA1) in breast cancer cells. a Upper: representitive western blots indicate the precipitation (IP) of IMP1 in T47D cells from co-IP experiments; lower: RT-qPCR shows that UCA1 was preferentially co-precipitated with IMP1 antibody relative to the IgG control. Data are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed: ** P

Techniques Used: RNA Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to urethral carcinoma-associated 1 (UCA1) and decreases UCA1 stability. a Upper: shows MS2-UCA1 fusion RNA. UCA1 with mutated IMP1 binding sites are shown by red arrow heads. Lower; UCA1 RNAs were pulled down by MBP-MCP; immunoblots show that interaction of IMP1 with UCA1 was greatly reduced when the IMP1 binding motifs were mutated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. b Stability of UCA1 in T47D and T47D-IMP1/short hairpin RNA (shRNA) cells were measured after treatment with actinomycin D for 12 h. Relative UCA1 levels were determined by normalizing to GAPDH messenger RNA (mRNA) levels. Data are presented as means ± SD from three independent experiments: ** P
Figure Legend Snippet: Insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to urethral carcinoma-associated 1 (UCA1) and decreases UCA1 stability. a Upper: shows MS2-UCA1 fusion RNA. UCA1 with mutated IMP1 binding sites are shown by red arrow heads. Lower; UCA1 RNAs were pulled down by MBP-MCP; immunoblots show that interaction of IMP1 with UCA1 was greatly reduced when the IMP1 binding motifs were mutated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. b Stability of UCA1 in T47D and T47D-IMP1/short hairpin RNA (shRNA) cells were measured after treatment with actinomycin D for 12 h. Relative UCA1 levels were determined by normalizing to GAPDH messenger RNA (mRNA) levels. Data are presented as means ± SD from three independent experiments: ** P

Techniques Used: RNA Binding Assay, Binding Assay, Western Blot, shRNA

15) Product Images from "Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes"

Article Title: Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes

Journal: Journal of Neuroinflammation

doi: 10.1186/s12974-014-0214-3

HIV-1 Tat-mediated expression of IL-6 and IL-8 involves PI3K/Akt pathway. (a-f) Astrocytes were pretreated with a specific PI3K inhibitor (LY294002) for 1 hour prior to transfection. (a, b) The expression levels of IL-6 and IL-8 at the level of mRNA were determined at 6 hours post transfection by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (c, d) IL-6 and IL-8 protein concentrations in the cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. (e) Astrocytes were either mock-transfected or transfected with HIV-1 Tat plasmid for duration of 6 hours and p-Akt levels were measured in whole cell extracts. (f) Astrocytes were transfected for a duration of 6 hours and translocation of p65 was measured. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and LaminB were used as internal loading controls for cytoplasmic and nuclear protein fractions, respectively. A representative Western blot is shown in figures (e) and (f) . (g-j) Astrocytes were transfected with either scrambled or Akt1 or Akt2 or Akt3 siRNA for a duration of 48 hours, followed by either mock transfection or transfection with HIV-1 Tat plasmid. (g, h) The levels of IL-6 and IL-8 at mRNA level were determined by real time RT-PCR at 6 hours post transfection. (i, j) The protein concentrations of IL-6 and IL-8 in cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. Statistical analyses was performed by one-way ANOVA and ** denotes P -value of ≤ 0.01 and * denotes P -value of ≤ 0.05.
Figure Legend Snippet: HIV-1 Tat-mediated expression of IL-6 and IL-8 involves PI3K/Akt pathway. (a-f) Astrocytes were pretreated with a specific PI3K inhibitor (LY294002) for 1 hour prior to transfection. (a, b) The expression levels of IL-6 and IL-8 at the level of mRNA were determined at 6 hours post transfection by real time RT-PCR. The values represented are normalized to their mock-transfected controls. (c, d) IL-6 and IL-8 protein concentrations in the cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. (e) Astrocytes were either mock-transfected or transfected with HIV-1 Tat plasmid for duration of 6 hours and p-Akt levels were measured in whole cell extracts. (f) Astrocytes were transfected for a duration of 6 hours and translocation of p65 was measured. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and LaminB were used as internal loading controls for cytoplasmic and nuclear protein fractions, respectively. A representative Western blot is shown in figures (e) and (f) . (g-j) Astrocytes were transfected with either scrambled or Akt1 or Akt2 or Akt3 siRNA for a duration of 48 hours, followed by either mock transfection or transfection with HIV-1 Tat plasmid. (g, h) The levels of IL-6 and IL-8 at mRNA level were determined by real time RT-PCR at 6 hours post transfection. (i, j) The protein concentrations of IL-6 and IL-8 in cell culture supernatants at 48 hours post transfection were determined by multiplex cytokine assay. Each experiment was done at least in triplicate and each bar represents the ± SE of three individual experiments. Statistical analyses was performed by one-way ANOVA and ** denotes P -value of ≤ 0.01 and * denotes P -value of ≤ 0.05.

Techniques Used: Expressing, Transfection, Quantitative RT-PCR, Cell Culture, Multiplex Assay, Cytokine Assay, Plasmid Preparation, Translocation Assay, Western Blot

16) Product Images from "1,8-Cineole Ameliorates Steatosis of Pten Liver Specific KO Mice via Akt Inactivation"

Article Title: 1,8-Cineole Ameliorates Steatosis of Pten Liver Specific KO Mice via Akt Inactivation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms160612051

RT-PCR of the liver messenger RNA in the two groups. ( a ) fatty acid synthase (FASN); ( b ) fibroblast growth factor 21 (FGF21); ( c ) collagen 1A1 (COL1A1); ( d ) Liver X Receptor alpha (LXR alpha); ( e ) ATP-binding cassette, sub-family A, member 1 (Abca1); and ( f ) Glucose 6 Phosphatase (G6P). Data shows relative expression normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * p
Figure Legend Snippet: RT-PCR of the liver messenger RNA in the two groups. ( a ) fatty acid synthase (FASN); ( b ) fibroblast growth factor 21 (FGF21); ( c ) collagen 1A1 (COL1A1); ( d ) Liver X Receptor alpha (LXR alpha); ( e ) ATP-binding cassette, sub-family A, member 1 (Abca1); and ( f ) Glucose 6 Phosphatase (G6P). Data shows relative expression normalized to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). * p

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Binding Assay, Expressing

( a ) Western blot of fatty acid synthase (FASN), phospho Akt (P-Akt), total Akt (T-Akt), phospho mTOR (P-mTOR), total mTOR (mTOR), Phospho-protein phosphatase type 2A (P-PP2A), total PP2A (PP2A), phospho-insulin receptor (P-insulin receptor), total insulin receptor (insulin receptor) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the 1,8-cineole group, FASN, P-Akt, P-mTOR, and P-PP2A were decreased. P-insulin receptor was increased in the 1,8-cineole group. Densitometry of FASN/GAPDH ( b ), phospho/total Akt ( c ), phospho/total mTOR ( d ), phospho/total PP2A ( e ), and phospho/total insulin receptor ( f ) in the two groups ( n = 3). In the 1,8-cineole group, Akt and PP2A were significantly dephosphorylated, and the insulin receptor was significantly activated. * p
Figure Legend Snippet: ( a ) Western blot of fatty acid synthase (FASN), phospho Akt (P-Akt), total Akt (T-Akt), phospho mTOR (P-mTOR), total mTOR (mTOR), Phospho-protein phosphatase type 2A (P-PP2A), total PP2A (PP2A), phospho-insulin receptor (P-insulin receptor), total insulin receptor (insulin receptor) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the 1,8-cineole group, FASN, P-Akt, P-mTOR, and P-PP2A were decreased. P-insulin receptor was increased in the 1,8-cineole group. Densitometry of FASN/GAPDH ( b ), phospho/total Akt ( c ), phospho/total mTOR ( d ), phospho/total PP2A ( e ), and phospho/total insulin receptor ( f ) in the two groups ( n = 3). In the 1,8-cineole group, Akt and PP2A were significantly dephosphorylated, and the insulin receptor was significantly activated. * p

Techniques Used: Western Blot

17) Product Images from "Effect of 15-Deoxy-∆12,14-prostaglandin J2 Nanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model"

Article Title: Effect of 15-Deoxy-∆12,14-prostaglandin J2 Nanocapsules on Inflammation and Bone Regeneration in a Rat Bone Defect Model

Journal: Chinese Medical Journal

doi: 10.4103/0366-6999.198924

Effects of 15d-PGJ2 on IL-6, IL-1β, and TNF-α protein expression in the peri-defect tissues. A 5 mm × 1.5 mm transcortical defect was prepared through the cortical bone into the bone marrow in the midshaft area in rats. Protein expression levels of IL-6, IL-1β, and TNF-α in groups treated with saline (S), empty nanocapsules (K), free 15d-PGJ2 (F), and 15d-PGJ2-NC (N) were analyzed by Western blotting on days 1, 3, 7, and 14. 15d-PGJ2-NC: 15-Deoxy-Δ12,14-prostaglandin J2 nanocapsules; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.
Figure Legend Snippet: Effects of 15d-PGJ2 on IL-6, IL-1β, and TNF-α protein expression in the peri-defect tissues. A 5 mm × 1.5 mm transcortical defect was prepared through the cortical bone into the bone marrow in the midshaft area in rats. Protein expression levels of IL-6, IL-1β, and TNF-α in groups treated with saline (S), empty nanocapsules (K), free 15d-PGJ2 (F), and 15d-PGJ2-NC (N) were analyzed by Western blotting on days 1, 3, 7, and 14. 15d-PGJ2-NC: 15-Deoxy-Δ12,14-prostaglandin J2 nanocapsules; IL-1β: Interleukin-1 beta; IL-6: Interleukin-6; TNF-α: Tumor necrosis factor-alpha; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Techniques Used: Expressing, Western Blot

18) Product Images from "CCR7 Is Important for Mesangial Cell Physiology and Repair"

Article Title: CCR7 Is Important for Mesangial Cell Physiology and Repair

Journal: Journal of Histochemistry and Cytochemistry

doi: 10.1369/0022155417737975

CCR7 protein expression by 10 and 20w WT and CCR7 –/– pMMCs. Ten µg protein from the respective pMMC lysate was used for SDS-PAGE. Murine CCR7 has a molecular weight of 43 kDa, GAPDH of 36 kDa. Abbreviations: WT, wild-type; pMMCs, primary mouse mesangial cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Figure Legend Snippet: CCR7 protein expression by 10 and 20w WT and CCR7 –/– pMMCs. Ten µg protein from the respective pMMC lysate was used for SDS-PAGE. Murine CCR7 has a molecular weight of 43 kDa, GAPDH of 36 kDa. Abbreviations: WT, wild-type; pMMCs, primary mouse mesangial cells; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Techniques Used: Expressing, SDS Page, Molecular Weight

19) Product Images from "INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR INHIBITOR, AMG-479, IN CETUXIMAB-REFRACTORY HEAD AND NECK SQUAMOUS CELL CARCINOMA"

Article Title: INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR INHIBITOR, AMG-479, IN CETUXIMAB-REFRACTORY HEAD AND NECK SQUAMOUS CELL CARCINOMA

Journal: Head & neck

doi: 10.1002/hed.21478

Western blot comparison of protein levels in the tumors before and after AMG-479 treatment. IGF-1R, insulin-like growth factor-1 receptor; AKT, protein kinase B; DHFR, dihydrofolate reductase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure Legend Snippet: Western blot comparison of protein levels in the tumors before and after AMG-479 treatment. IGF-1R, insulin-like growth factor-1 receptor; AKT, protein kinase B; DHFR, dihydrofolate reductase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Techniques Used: Western Blot

20) Product Images from "Pressure Overload-Induced Cardiac Hypertrophy Response Requires Janus Kinase 2-Histone Deacetylase 2 Signaling"

Article Title: Pressure Overload-Induced Cardiac Hypertrophy Response Requires Janus Kinase 2-Histone Deacetylase 2 Signaling

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms151120240

Janus kinase 2 (Jak2) and histone deacetylase 2 (HDAC2) are important for angiotensin II (Ang-II)-induced atrial natriuretic peptide ( ANP )/brain natriuretic peptide ( BNP ) expression in H9c2 cardiomyocytes. Protein and mRNA expression of HDAC2 ( A , B ) or Jak2 ( C , D ) in H9c2 cells transfected with scramble siRNA (sc-siRNA) or indicated siRNA (100 nM each, 48 h); Protein ( E ) and mRNA ( F , G ) expression of ANP , BNP and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) before and after Ang-II (1 μM, 24 h) treatment in H9c2 cells transfected with indicated siRNA; Protein ( H ) and mRNA expression of HDAC2 ( H , I ) and Jak2 ( J , K ) in stable H9c2 cells transfected with vector (“Vec”) or indicated cDNA plasmid (0.5 μg/mL each); Message RNA (mRNA) expression of ANP ( L ) and BNP ( M ) before and after Ang-II (1 μM, 24 h) treatment in stable H9c2 cells transfected with Jak2/HDAC2 plasmid or empty vector (“Vec”, 0.5 μg/mL each). For all the mRNA assays, n = 6. Experiments in this figure were repeated three times with similar results obtained. All data were expressed as means ± SEM, GAPDH was tested as an internal control. * p
Figure Legend Snippet: Janus kinase 2 (Jak2) and histone deacetylase 2 (HDAC2) are important for angiotensin II (Ang-II)-induced atrial natriuretic peptide ( ANP )/brain natriuretic peptide ( BNP ) expression in H9c2 cardiomyocytes. Protein and mRNA expression of HDAC2 ( A , B ) or Jak2 ( C , D ) in H9c2 cells transfected with scramble siRNA (sc-siRNA) or indicated siRNA (100 nM each, 48 h); Protein ( E ) and mRNA ( F , G ) expression of ANP , BNP and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) before and after Ang-II (1 μM, 24 h) treatment in H9c2 cells transfected with indicated siRNA; Protein ( H ) and mRNA expression of HDAC2 ( H , I ) and Jak2 ( J , K ) in stable H9c2 cells transfected with vector (“Vec”) or indicated cDNA plasmid (0.5 μg/mL each); Message RNA (mRNA) expression of ANP ( L ) and BNP ( M ) before and after Ang-II (1 μM, 24 h) treatment in stable H9c2 cells transfected with Jak2/HDAC2 plasmid or empty vector (“Vec”, 0.5 μg/mL each). For all the mRNA assays, n = 6. Experiments in this figure were repeated three times with similar results obtained. All data were expressed as means ± SEM, GAPDH was tested as an internal control. * p

Techniques Used: Histone Deacetylase Assay, Aqueous Normal-phase Chromatography, Expressing, Transfection, Plasmid Preparation

21) Product Images from "Vitamin D maintains E-cadherin intercellular junctions by downregulating MMP-9 production in human gingival keratinocytes treated by TNF-α"

Article Title: Vitamin D maintains E-cadherin intercellular junctions by downregulating MMP-9 production in human gingival keratinocytes treated by TNF-α

Journal: Journal of Periodontal & Implant Science

doi: 10.5051/jpis.2019.49.5.270

Vitamin D inhibits TNF-α-induced NF-κB signaling, as shown by western blotting. Cells were cultured for 48 hours and then pretreated with vitamin D (10, 100, or 1,000 nM) or without it for 24 hours. Before cell lysis for western blotting, cells were treated with TNF-α (100 ng/mL) for 10 minutes. Bay 11-7082 (Bay 11; 1 nM), a pharmacological inactivator of NF-κB signaling, was used as an internal control of the immunoblotting analyses to show that TNF-α-induced NF-κB signaling was reduced when NF-κB signaling was pharmacologically inactivated in HGKs. TNF-α: tumor necrosis factor-alpha, NF-κB: nuclear factor kappa B, HGK: human gingival keratinocyte, GAPDH: glyceraldehyde 3-phosphate dehydrogenase.
Figure Legend Snippet: Vitamin D inhibits TNF-α-induced NF-κB signaling, as shown by western blotting. Cells were cultured for 48 hours and then pretreated with vitamin D (10, 100, or 1,000 nM) or without it for 24 hours. Before cell lysis for western blotting, cells were treated with TNF-α (100 ng/mL) for 10 minutes. Bay 11-7082 (Bay 11; 1 nM), a pharmacological inactivator of NF-κB signaling, was used as an internal control of the immunoblotting analyses to show that TNF-α-induced NF-κB signaling was reduced when NF-κB signaling was pharmacologically inactivated in HGKs. TNF-α: tumor necrosis factor-alpha, NF-κB: nuclear factor kappa B, HGK: human gingival keratinocyte, GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Techniques Used: Western Blot, Cell Culture, Lysis

22) Product Images from "PRSS contributes to cetuximab resistance in colorectal cancer"

Article Title: PRSS contributes to cetuximab resistance in colorectal cancer

Journal: Science Advances

doi: 10.1126/sciadv.aax5576

PRSS1 leads to cetuximab resistance. ( A ) Heat map representation of PRSS gene expression ( PRSS1 , PRSS2 , and PRSS3 ) in a panel of cetuximab-resistant cell lines ( n = 19) and cetuximab-sensitive cell lines ( n = 30). Gene clustering was performed with Euclidean distance as a similarity metric. Values are log 2 median-centered intensities. ( B ) RT-PCR and Western blot measurements of the expression of PRSS family genes in a panel of colon cancer cell lines ( n = 6). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( C ) Real-time PCR measurement of relative PRSS1 expression in a panel of colon cancer cell lines ( n = 6). Data shown are the means ± SD of triplicate measurements that had been repeated three times with similar results. ( D ) ELISA measurement of PRSS1 expression in a panel of colon cancer cell lines ( n = 6). Data shown are the means ± SD of triplicate measurements that had been repeated three times with similar results. ( E ) Left: Representative IHC staining of PRSS1 in human CRC samples. Scale bar, 100 μm. Right: Correlation of cetuximab effectiveness (response or resistance) with positive PRSS1 staining. To quantify positive PRSS1 staining, images were taken from eight areas per tissue sample. Differences in growth were determined using Student’s t test and by calculating subsequent P values. *** P
Figure Legend Snippet: PRSS1 leads to cetuximab resistance. ( A ) Heat map representation of PRSS gene expression ( PRSS1 , PRSS2 , and PRSS3 ) in a panel of cetuximab-resistant cell lines ( n = 19) and cetuximab-sensitive cell lines ( n = 30). Gene clustering was performed with Euclidean distance as a similarity metric. Values are log 2 median-centered intensities. ( B ) RT-PCR and Western blot measurements of the expression of PRSS family genes in a panel of colon cancer cell lines ( n = 6). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. ( C ) Real-time PCR measurement of relative PRSS1 expression in a panel of colon cancer cell lines ( n = 6). Data shown are the means ± SD of triplicate measurements that had been repeated three times with similar results. ( D ) ELISA measurement of PRSS1 expression in a panel of colon cancer cell lines ( n = 6). Data shown are the means ± SD of triplicate measurements that had been repeated three times with similar results. ( E ) Left: Representative IHC staining of PRSS1 in human CRC samples. Scale bar, 100 μm. Right: Correlation of cetuximab effectiveness (response or resistance) with positive PRSS1 staining. To quantify positive PRSS1 staining, images were taken from eight areas per tissue sample. Differences in growth were determined using Student’s t test and by calculating subsequent P values. *** P

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

23) Product Images from "IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p"

Article Title: IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p

Journal: Breast Cancer Research : BCR

doi: 10.1186/s13058-018-0959-1

Differential expression of selected microarray-identified long non-coding RNAs (lncRNAs) and their binding to insulin-like growth factor 2 messenger RNA binding protein (IMP1). a Total RNA was extracted from MDA231 cells expressing green fluorescent protein (GFP) or Flag-tagged IMP1-GFP. RT-qPCR was used to analyze the levels of six microarray-identified lncRNAs. Relative levels of the lncRNAs were nomalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed. The data are presented as means ± SD from three independent experiments: * P
Figure Legend Snippet: Differential expression of selected microarray-identified long non-coding RNAs (lncRNAs) and their binding to insulin-like growth factor 2 messenger RNA binding protein (IMP1). a Total RNA was extracted from MDA231 cells expressing green fluorescent protein (GFP) or Flag-tagged IMP1-GFP. RT-qPCR was used to analyze the levels of six microarray-identified lncRNAs. Relative levels of the lncRNAs were nomalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed. The data are presented as means ± SD from three independent experiments: * P

Techniques Used: Expressing, Microarray, Binding Assay, RNA Binding Assay, Quantitative RT-PCR

Interaction of insulin-like growth factor 2 messenger RNA binding protein (IMP1) with urethral carcinoma-associated 1 (UCA1) in breast cancer cells. a Upper: representitive western blots indicate the precipitation (IP) of IMP1 in T47D cells from co-IP experiments; lower: RT-qPCR shows that UCA1 was preferentially co-precipitated with IMP1 antibody relative to the IgG control. Data are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed: ** P
Figure Legend Snippet: Interaction of insulin-like growth factor 2 messenger RNA binding protein (IMP1) with urethral carcinoma-associated 1 (UCA1) in breast cancer cells. a Upper: representitive western blots indicate the precipitation (IP) of IMP1 in T47D cells from co-IP experiments; lower: RT-qPCR shows that UCA1 was preferentially co-precipitated with IMP1 antibody relative to the IgG control. Data are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) and statistically analyzed: ** P

Techniques Used: RNA Binding Assay, Western Blot, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to urethral carcinoma-associated 1 (UCA1) and decreases UCA1 stability. a Upper: shows MS2-UCA1 fusion RNA. UCA1 with mutated IMP1 binding sites are shown by red arrow heads. Lower; UCA1 RNAs were pulled down by MBP-MCP; immunoblots show that interaction of IMP1 with UCA1 was greatly reduced when the IMP1 binding motifs were mutated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. b Stability of UCA1 in T47D and T47D-IMP1/short hairpin RNA (shRNA) cells were measured after treatment with actinomycin D for 12 h. Relative UCA1 levels were determined by normalizing to GAPDH messenger RNA (mRNA) levels. Data are presented as means ± SD from three independent experiments: ** P
Figure Legend Snippet: Insulin-like growth factor 2 messenger RNA binding protein (IMP1) binds to urethral carcinoma-associated 1 (UCA1) and decreases UCA1 stability. a Upper: shows MS2-UCA1 fusion RNA. UCA1 with mutated IMP1 binding sites are shown by red arrow heads. Lower; UCA1 RNAs were pulled down by MBP-MCP; immunoblots show that interaction of IMP1 with UCA1 was greatly reduced when the IMP1 binding motifs were mutated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a control. b Stability of UCA1 in T47D and T47D-IMP1/short hairpin RNA (shRNA) cells were measured after treatment with actinomycin D for 12 h. Relative UCA1 levels were determined by normalizing to GAPDH messenger RNA (mRNA) levels. Data are presented as means ± SD from three independent experiments: ** P

Techniques Used: RNA Binding Assay, Binding Assay, Western Blot, shRNA

24) Product Images from "Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer"

Article Title: Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3063

Combination of α-TEA + TAM acts cooperatively to reduce prosurvival signaling in TAMR cells . (a, b) Western immunoblot analyses using aliquots of cell lysates from treated cells in Figure 3b were performed to assess prosurvival signaling mediators (a) and to assess antiapoptotic factors c-FLIP and Bcl-2 protein expression (b). (c) MCF-7/TAMR cells transfected with siRNAs to Akt-1 or c-FLIP, as well as control siRNA (labeled Control), were treated with α-TEA (20 μ M ) for 1 day. Western immunoblot analyses were performed to determine PARP, pJNK2/1, CHOP, DR5 (L/S), GRP 78, pAKT, and c-FLIP protein levels, with GAPDH serving as loading control. (a-c) Data are representative of three individual experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; Bcl-2, B-cell lymphoma 2; c-FLIP, cellular FLICE-inhibitory protein; CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; pJNK, phosphorylated-c-Jun N-terminal kinase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant.
Figure Legend Snippet: Combination of α-TEA + TAM acts cooperatively to reduce prosurvival signaling in TAMR cells . (a, b) Western immunoblot analyses using aliquots of cell lysates from treated cells in Figure 3b were performed to assess prosurvival signaling mediators (a) and to assess antiapoptotic factors c-FLIP and Bcl-2 protein expression (b). (c) MCF-7/TAMR cells transfected with siRNAs to Akt-1 or c-FLIP, as well as control siRNA (labeled Control), were treated with α-TEA (20 μ M ) for 1 day. Western immunoblot analyses were performed to determine PARP, pJNK2/1, CHOP, DR5 (L/S), GRP 78, pAKT, and c-FLIP protein levels, with GAPDH serving as loading control. (a-c) Data are representative of three individual experiments. α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue; Bcl-2, B-cell lymphoma 2; c-FLIP, cellular FLICE-inhibitory protein; CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; pJNK, phosphorylated-c-Jun N-terminal kinase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant.

Techniques Used: Western Blot, Expressing, Transfection, Labeling, Binding Assay, Small Interfering RNA

α-TEA alone and in combination with TAM induces biomarkers of endoplasmic reticulum stress, and siRNA knockdowns show necessary roles for CHOP, DR5, and JNK . (a) Western immunoblot analyses, using aliquots of cell lysates from cells treated with 20 μ M α-TEA and 1 μ M TAM in Figure 3b, were performed to assess pJNK2/1, CHOP, and DR5 (L/S) protein levels, as well as endoplasmic reticulum stress marker GRP78 protein expression with GAPDH as loading control. (b) MCF-7/TAMR cells transfected with siRNAs to CHOP, DR5, and JNK as well as control siRNA (labeled Control) were treated with TAM (1 μ M ) + α-TEA (20 μM) for 1 day. Western immunoblot analyses were performed to determine degree of apoptosis, as measured by cleaved PARP (cPARP), pJNK2/1, CHOP, and DR5 (L/S) protein levels, with GAPDH serving as the loading control. Data from (a) and (b) are representative of three individual experiments. CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; JNK, c-Jun N-terminal kinase; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant; α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue.
Figure Legend Snippet: α-TEA alone and in combination with TAM induces biomarkers of endoplasmic reticulum stress, and siRNA knockdowns show necessary roles for CHOP, DR5, and JNK . (a) Western immunoblot analyses, using aliquots of cell lysates from cells treated with 20 μ M α-TEA and 1 μ M TAM in Figure 3b, were performed to assess pJNK2/1, CHOP, and DR5 (L/S) protein levels, as well as endoplasmic reticulum stress marker GRP78 protein expression with GAPDH as loading control. (b) MCF-7/TAMR cells transfected with siRNAs to CHOP, DR5, and JNK as well as control siRNA (labeled Control) were treated with TAM (1 μ M ) + α-TEA (20 μM) for 1 day. Western immunoblot analyses were performed to determine degree of apoptosis, as measured by cleaved PARP (cPARP), pJNK2/1, CHOP, and DR5 (L/S) protein levels, with GAPDH serving as the loading control. Data from (a) and (b) are representative of three individual experiments. CHOP, Ccaat-enhancer-binding protein (C/EBP) homologous protein; DR5 (L/S), death receptor 5 long/short; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRP78, glucose-regulated protein-78; JNK, c-Jun N-terminal kinase; MCF-7/TAMR, acquired tamoxifen-resistant MCF-7; PARP, poly (ADP-ribose) polymerase; siRNA, small interfering RNA; TAM, tamoxifen; TAMR, tamoxifen resistant; α-TEA, RRR-α-tocopherol ether-linked acetic acid analogue.

Techniques Used: Western Blot, Marker, Expressing, Transfection, Labeling, Binding Assay, Small Interfering RNA

25) Product Images from "Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin’s glucose-lowering effects"

Article Title: Endothelial APLNR regulates tissue fatty acid uptake and is essential for apelin’s glucose-lowering effects

Journal: Science translational medicine

doi: 10.1126/scitranslmed.aad4000

Endothelial APLNR is critical for apelin signaling ( A ) Representative immunoblot of AKT and AMPKα phosphorylation in response to apelin stimulation in skeletal muscle with or without EC depletion ( n = 3 replicates per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.s., not significant. ( B ) Representative immunoblot of AKT, AMPKα, and eNOS phosphorylation in response to apelin stimulation in skeletal muscle of Aplnr ECKO mice and control littermates ( n = 3 replicates per group). ( C ) IPGTT of Aplnr ECKO mice and control littermates [ n = 10 (control) and 14 ( Aplnr ECKO )]. ( D ) IPGTT in Aplnr ECKO and control mice 30 min after intravenous apelin injection [ n = 5 (control) and 7 ( Aplnr ECKO )]. ( E ) Intraperitoneal insulin tolerance testing of Aplnr ECKO mice and their control littermates under basal conditions ( n = 6 per group). ( F ) Representative apelin expression in skeletal muscle, WAT, and BAT, as assessed by lacZ staining of Apln +/lacz reporter mice fed a normal chow or a high-fat diet (HFD). Scale bar, 100 µm. ( G ) Relative mRNA expression of apelin in various tissues of mice fed a normal chow or a high-fat diet ( n = 5 per group). * P
Figure Legend Snippet: Endothelial APLNR is critical for apelin signaling ( A ) Representative immunoblot of AKT and AMPKα phosphorylation in response to apelin stimulation in skeletal muscle with or without EC depletion ( n = 3 replicates per group). GAPDH, glyceraldehyde-3-phosphate dehydrogenase; n.s., not significant. ( B ) Representative immunoblot of AKT, AMPKα, and eNOS phosphorylation in response to apelin stimulation in skeletal muscle of Aplnr ECKO mice and control littermates ( n = 3 replicates per group). ( C ) IPGTT of Aplnr ECKO mice and control littermates [ n = 10 (control) and 14 ( Aplnr ECKO )]. ( D ) IPGTT in Aplnr ECKO and control mice 30 min after intravenous apelin injection [ n = 5 (control) and 7 ( Aplnr ECKO )]. ( E ) Intraperitoneal insulin tolerance testing of Aplnr ECKO mice and their control littermates under basal conditions ( n = 6 per group). ( F ) Representative apelin expression in skeletal muscle, WAT, and BAT, as assessed by lacZ staining of Apln +/lacz reporter mice fed a normal chow or a high-fat diet (HFD). Scale bar, 100 µm. ( G ) Relative mRNA expression of apelin in various tissues of mice fed a normal chow or a high-fat diet ( n = 5 per group). * P

Techniques Used: Mouse Assay, Injection, Expressing, Staining

26) Product Images from "Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells"

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19051498

Effect of transforming growth factor β (TGFβ 2 ) on SK-1, phospho-Smad2, connective tissue growth factor (CTGF), and fibronectin expression in HK2 cells. ( A ) HK2 cells were taken unstimulated (−) or stimulated with 5 ng/mL of TGFβ 2 for the indicated time periods up to 24 h. Thereafter, cell lysates were prepared and equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and subjected to Western blotting using antibodies against SK-1 (1:2000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000), phospho-Smad2 (1:1000), CTGF (1:500), and fibronectin (1:500). To detect secreted CTGF, equal volumes of supernatants of stimulated cells were taken for protein precipitation by trichloroacetic acid (TCA) and were then analysed the same way as described for lysates using the CTGF antibody. Band intensities were evaluated by a LICOR R imaging system. Data in ( B – F ) show the evaluated respective bands. Results are expressed as fold increase and are means ± S.D. ( n = 3); * p
Figure Legend Snippet: Effect of transforming growth factor β (TGFβ 2 ) on SK-1, phospho-Smad2, connective tissue growth factor (CTGF), and fibronectin expression in HK2 cells. ( A ) HK2 cells were taken unstimulated (−) or stimulated with 5 ng/mL of TGFβ 2 for the indicated time periods up to 24 h. Thereafter, cell lysates were prepared and equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and subjected to Western blotting using antibodies against SK-1 (1:2000 dilution), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000), phospho-Smad2 (1:1000), CTGF (1:500), and fibronectin (1:500). To detect secreted CTGF, equal volumes of supernatants of stimulated cells were taken for protein precipitation by trichloroacetic acid (TCA) and were then analysed the same way as described for lysates using the CTGF antibody. Band intensities were evaluated by a LICOR R imaging system. Data in ( B – F ) show the evaluated respective bands. Results are expressed as fold increase and are means ± S.D. ( n = 3); * p

Techniques Used: Expressing, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Imaging

27) Product Images from "Paeonol Ameliorates Ovalbumin-Induced Asthma through the Inhibition of TLR4/NF-κB and MAPK Signaling"

Article Title: Paeonol Ameliorates Ovalbumin-Induced Asthma through the Inhibition of TLR4/NF-κB and MAPK Signaling

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2018/3063145

Changed expression of p-P38 and p-ERK in asthma and paeonol treatment. (a) The expression of p-ERK, ERK, p-P38, and P38 was analysed by immune blot. GAPDH was utilized as the standard control. (b) The band signal strengths of p-ERK and ERK were expressed as a ratio to ERK and GAPDH, respectively (n= 4 per group). (c) The band signal strengths of p-P38 and P38 were expressed as a ratio to P38 and GAPDH, respectively. The data were expressed as the means ± SEM (n = 4 per group). Extracellular signal-regulated kinase, ERK; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; ovalbumin, OVA; control, Con; montelukast sodium, Mon. ∗ P
Figure Legend Snippet: Changed expression of p-P38 and p-ERK in asthma and paeonol treatment. (a) The expression of p-ERK, ERK, p-P38, and P38 was analysed by immune blot. GAPDH was utilized as the standard control. (b) The band signal strengths of p-ERK and ERK were expressed as a ratio to ERK and GAPDH, respectively (n= 4 per group). (c) The band signal strengths of p-P38 and P38 were expressed as a ratio to P38 and GAPDH, respectively. The data were expressed as the means ± SEM (n = 4 per group). Extracellular signal-regulated kinase, ERK; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; ovalbumin, OVA; control, Con; montelukast sodium, Mon. ∗ P

Techniques Used: Expressing

28) Product Images from "Simultaneous blockade of interacting CK2 and EGFR pathways by tumor-targeting nanobioconjugates increases therapeutic efficacy against glioblastoma multiforme"

Article Title: Simultaneous blockade of interacting CK2 and EGFR pathways by tumor-targeting nanobioconjugates increases therapeutic efficacy against glioblastoma multiforme

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2016.11.001

Expression of molecular targets in xenogeneic LN229 brain tumors revealed by western blot. A) Protein expression of EGFR, CK2α, phosphorylated Akt [Ser473] (pAkt S473), total Akt, c-Myc, phosphorylated Cdc37 (pCdc37), and programmed death-ligand 1 (PD-L1). Treatment with all nanobioconjugates resulted in significantly lower expression of the analyzed proteins. Three independent tumor specimens per each treatment group were analyzed. AON - antisense oligonucleotide, PBS - phosphate-buffered saline, Cetu - cetuximab, CK2α - catalytic α subunit of protein kinase CK2, EGFR - epidermal growth factor receptor, P/Cetu/CK2α - nanobioconjugate with AON against CK2α, P/Cetu/EGFR - nanobioconjugate with AON against EGFR, P/Cetu/EGFR/CK2α - nanobioconjugate with AONs against both EGFR and CK2α, GAPDH - glyceraldehyde 3-phosphate dehydrogenase. B) Band intensities of tumor samples were quantified by Image Studio software and normalized against GAPDH or Akt (for pAkt) as a control. All proteins showed statistically significant decreases following the treatments. Note significantly reduced expression of EGFR upon anti-CK2α treatment and vice versa. The results represent means ± SEMs vs. PBS treatment. *, P
Figure Legend Snippet: Expression of molecular targets in xenogeneic LN229 brain tumors revealed by western blot. A) Protein expression of EGFR, CK2α, phosphorylated Akt [Ser473] (pAkt S473), total Akt, c-Myc, phosphorylated Cdc37 (pCdc37), and programmed death-ligand 1 (PD-L1). Treatment with all nanobioconjugates resulted in significantly lower expression of the analyzed proteins. Three independent tumor specimens per each treatment group were analyzed. AON - antisense oligonucleotide, PBS - phosphate-buffered saline, Cetu - cetuximab, CK2α - catalytic α subunit of protein kinase CK2, EGFR - epidermal growth factor receptor, P/Cetu/CK2α - nanobioconjugate with AON against CK2α, P/Cetu/EGFR - nanobioconjugate with AON against EGFR, P/Cetu/EGFR/CK2α - nanobioconjugate with AONs against both EGFR and CK2α, GAPDH - glyceraldehyde 3-phosphate dehydrogenase. B) Band intensities of tumor samples were quantified by Image Studio software and normalized against GAPDH or Akt (for pAkt) as a control. All proteins showed statistically significant decreases following the treatments. Note significantly reduced expression of EGFR upon anti-CK2α treatment and vice versa. The results represent means ± SEMs vs. PBS treatment. *, P

Techniques Used: Expressing, Western Blot, Software

Related Articles

MTT Assay:

Article Title: Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells
Article Snippet: Hoechst 33342 stain, 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), propidium iodide (PI), 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-imidacarbocyanine iodide (JC-1), and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) were purchased from Sigma-Aldrich (St. Louis, USA). .. Specific antibodies against Bcl-2, bcl-X protein (Bcl-XL), bcl-2-associated X protein (BAX), bcl-2 homologous antagonist-killer protein (BAK), poly-ADP-ribose polymerase (PARP), caspase-3, caspase-9, phospho-c-Jun N-terminal protein kinase (p-JNK), phospho-extracellular regulated protein kinase 1/2 (p-ERK), phospho-p38 mitogen-activated protein kinase (p-p38), JNK, p38, ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and related secondary antibodies were purchased from Cell Signaling Technology (Beverly, USA).

BIA-KA:

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages
Article Snippet: The concentrations of proteins in the extracts were determined using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). .. Each membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies against IL-1β , Iκ Bα , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IKKβ (Ser177/181), IKKα /β , phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, NLRP3, caspase-8 (Cell Signaling Technology, Danvers, MA, USA), pro-IL-18 (Medical & Biological Laboratories), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase-1 p20 (AdipoGen, San Diego, CA, USA) at a dilution of 1/1,000.

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Protein concentration was quantified using the Pierce BCA Protein Assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). .. Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK).

Pyrolysis Gas Chromatography:

Article Title: Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation
Article Snippet: .. Western blot analysis Protein lysates from cells were subjected to SDS-PAGE and target proteins were detected with primary antibodies recognizing p-p38, p38, P-extracellular signal-regulated kinase (P-ERK), ERK, p-JNK, JNK, p-NF-κB p65 (Ser536), NF-κβ p65 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST, Danvers, Massachusetts, USA), PGC-1β and RANKL (Epitomics, San Francisco, USA), MMP-3 and MMP-13 (Abcam, Cambridge, USA) respectively. .. After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (EarthOx, Millbrae, California, USA), protein bands were visualized using enhanced chemiluminescence (Millipore, Boston, USA) plus western blot detection reagents followed by exposure to a scanning imager (G:BOX Gel & Blot Imaging Series from Syngene, Cambridge, UK).

Construct:

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells
Article Snippet: .. S1P and all C17-sphingolipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL, USA); caged-S1P was from Enzo Life Sciences Inc. (Farmingdale, NY, USA); human TGF-β2 , TNFα, and IL-1β were from Peprotech (London, UK); BAF312 was from Selleck Chemicals Inc. (Houston, TX, USA); CYM5442, CYM5520, CYM5541, puromycin, the lentiviral particles of human Spns2 and SK-1 shRNA constructs (MISSION® shRNA), diamidine-phenylindole (DAPI), and the antibodies against α-tubulin and Spns2 (SAB2104271) were from Sigma Aldrich Chemikalien, (Buchs, Switzerland); antibodies against phospho-ERK1/2, phospho-Smad2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against CTGF (L-20) and fibronectin (EP5) were from Santa Cruz (Heidelberg, Germany); the AQP1 antibody ( (B)) was from Abcam plc (Cambridge, UK); the human SK-1 antibody was generated and characterized, as previously described [ , ]. .. The secondary anti-rabbit and anti-mouse horseradish peroxidase-coupled IgG antibodies and the enhanced chemiluminescence (ECL) reagents were from GE Health Care Systems GmbH (Freiburg, Germany).

Enzyme-linked Immunosorbent Assay:

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells
Article Snippet: S1P and all C17-sphingolipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL, USA); caged-S1P was from Enzo Life Sciences Inc. (Farmingdale, NY, USA); human TGF-β2 , TNFα, and IL-1β were from Peprotech (London, UK); BAF312 was from Selleck Chemicals Inc. (Houston, TX, USA); CYM5442, CYM5520, CYM5541, puromycin, the lentiviral particles of human Spns2 and SK-1 shRNA constructs (MISSION® shRNA), diamidine-phenylindole (DAPI), and the antibodies against α-tubulin and Spns2 (SAB2104271) were from Sigma Aldrich Chemikalien, (Buchs, Switzerland); antibodies against phospho-ERK1/2, phospho-Smad2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against CTGF (L-20) and fibronectin (EP5) were from Santa Cruz (Heidelberg, Germany); the AQP1 antibody ( (B)) was from Abcam plc (Cambridge, UK); the human SK-1 antibody was generated and characterized, as previously described [ , ]. .. The human MCP-1 ELISA was from Boster Biological Technology (Pleasanton, CA, USA).

Quantitation Assay:

Article Title: Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition
Article Snippet: Antibodies against SPARC, collagen 1A1 (COL1A1), FN1, MMP-14 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signalling Technology (Massachusetts, USA), Abnova Corp. (Colorado, USA), Epitomics (Abcam, Massachusetts, USA), Abcam (Massachusetts, USA) and Santa Cruz Biotechnology (California, USA), respectively. .. Densitometric quantitation was performed using Image Studio Lite V.5 (LI-COR Biosciences, Nevada, USA).

Article Title: Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells
Article Snippet: Antibodies specific for phospho-Src family (Tyr416) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). .. For signal quantitation, the bands were digitalized and analyzed by ImageJ software.

Expressing:

Article Title: Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes
Article Snippet: HIV-1 Tat expression plasmid, initially developed by Dr. E Verdin, Gladstone institute, UCSF (catalog # 10453), and HIV-1 Tat protein (catalog # 2222) were obtained from the NIH AIDS reagent program. .. The primary antibodies for p65, p-c-jun, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and all the secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA) and primary antibodies for p-p38, p-Akt, p-JNK and LaminB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK). .. Protein expression was detected with Super signal west pico chemilumninescent reagents (Thermo Scientific, Rockford, IL, USA) and quantified using Image lab TM Analyzer software (Bio-Rad laboratories N.V.).

Bradford Assay:

Article Title: Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B
Article Snippet: Protein concentrations of extracts from mouse tissues were isolated and extracted as previously described., Protein content was determined using BSA as a standard according to the Bradford assay. .. Blots were probed with antibodies against dysferlin (1:2,500, mouse monoclonal antibody), TRIM72/MG53 (1:2,500, custom rabbit polyclonal antibody), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000, D16H11; Cell Signaling), and appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology).

Western Blot:

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages
Article Snippet: Paragraph title: 2.5. Western Blotting ... Each membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies against IL-1β , Iκ Bα , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IKKβ (Ser177/181), IKKα /β , phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, NLRP3, caspase-8 (Cell Signaling Technology, Danvers, MA, USA), pro-IL-18 (Medical & Biological Laboratories), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase-1 p20 (AdipoGen, San Diego, CA, USA) at a dilution of 1/1,000.

Article Title: Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B
Article Snippet: Paragraph title: Western Blotting ... Blots were probed with antibodies against dysferlin (1:2,500, mouse monoclonal antibody), TRIM72/MG53 (1:2,500, custom rabbit polyclonal antibody), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000, D16H11; Cell Signaling), and appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology).

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Paragraph title: 4.5. Quantification of Myocardial Protein Levels by Western Blot ... Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK).

Article Title: Anorexic response to rapamycin does not appear to involve a central mechanism
Article Snippet: Inhibition of mTOR signalling was assessed by the amount of phosphorylated S6 (pS6) protein in the liver, MBH and HB by Western blot analysis. .. The pS6 antibody (4856S, Cell Signalling, Danvers, MA, USA) and total S6 antibody (2217S, Cell Signalling) were used in 1/1000; while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (3683S, Cell Signalling) antibody was used in 1/10000 concentrations for normalization.

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats
Article Snippet: Paragraph title: Cell Culture and Western Blot Analysis ... The membranes were blocked in 5% dry milk for 1 h and incubated overnight with one of the following primary antibodies at 4°C: total AKT (1∶1000, Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Ser1177 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Thr495 (1∶500, Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Cell Signaling Technology, Danvers, MA, USA), and Mas (1∶500) .

Article Title: Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation
Article Snippet: .. Western blot analysis Protein lysates from cells were subjected to SDS-PAGE and target proteins were detected with primary antibodies recognizing p-p38, p38, P-extracellular signal-regulated kinase (P-ERK), ERK, p-JNK, JNK, p-NF-κB p65 (Ser536), NF-κβ p65 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST, Danvers, Massachusetts, USA), PGC-1β and RANKL (Epitomics, San Francisco, USA), MMP-3 and MMP-13 (Abcam, Cambridge, USA) respectively. .. After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (EarthOx, Millbrae, California, USA), protein bands were visualized using enhanced chemiluminescence (Millipore, Boston, USA) plus western blot detection reagents followed by exposure to a scanning imager (G:BOX Gel & Blot Imaging Series from Syngene, Cambridge, UK).

Protease Inhibitor:

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages
Article Snippet: Western Blotting Whole cellular proteins were extracted using RIPA Buffer (Sigma-Aldrich) supplemented with the Complete Protease Inhibitor Cocktail Tablet (Roche Life Science) and Phosphatase Inhibitor Cocktail (Nacalai Tesque). .. Each membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies against IL-1β , Iκ Bα , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IKKβ (Ser177/181), IKKα /β , phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, NLRP3, caspase-8 (Cell Signaling Technology, Danvers, MA, USA), pro-IL-18 (Medical & Biological Laboratories), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase-1 p20 (AdipoGen, San Diego, CA, USA) at a dilution of 1/1,000.

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Tissues were placed in lysing matrix tubes (QBiogene/MP Biomedicals, Solon, OH, USA), mixed with 1 mL of protein extraction buffer containing 10 mM imidazole, 300 mM sucrose, 1 mM dithiotreitol, 1mM sodium metabisulfite, 25 mM sodium fluoride, 5 mM sodium ethylenediaminetetraacetic acid, 5 mM sodium pyrophosphate, 0.3 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche Diagnostics Belgium, Vilvoorde, Belgium) (Lenaerts et al., 2013), and homogenized in the FastPrep24 instrument (MP Biomedicals). .. Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK).

Cell Culture:

Article Title: Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes
Article Snippet: Paragraph title: Cell culture and reagents ... The primary antibodies for p65, p-c-jun, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and all the secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA) and primary antibodies for p-p38, p-Akt, p-JNK and LaminB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells
Article Snippet: S1P and all C17-sphingolipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL, USA); caged-S1P was from Enzo Life Sciences Inc. (Farmingdale, NY, USA); human TGF-β2 , TNFα, and IL-1β were from Peprotech (London, UK); BAF312 was from Selleck Chemicals Inc. (Houston, TX, USA); CYM5442, CYM5520, CYM5541, puromycin, the lentiviral particles of human Spns2 and SK-1 shRNA constructs (MISSION® shRNA), diamidine-phenylindole (DAPI), and the antibodies against α-tubulin and Spns2 (SAB2104271) were from Sigma Aldrich Chemikalien, (Buchs, Switzerland); antibodies against phospho-ERK1/2, phospho-Smad2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against CTGF (L-20) and fibronectin (EP5) were from Santa Cruz (Heidelberg, Germany); the AQP1 antibody ( (B)) was from Abcam plc (Cambridge, UK); the human SK-1 antibody was generated and characterized, as previously described [ , ]. .. All of the cell culture nutrients were from Life Technologies AG (Basel, Switzerland).

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats
Article Snippet: Paragraph title: Cell Culture and Western Blot Analysis ... The membranes were blocked in 5% dry milk for 1 h and incubated overnight with one of the following primary antibodies at 4°C: total AKT (1∶1000, Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Ser1177 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Thr495 (1∶500, Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Cell Signaling Technology, Danvers, MA, USA), and Mas (1∶500) .

Generated:

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells
Article Snippet: .. S1P and all C17-sphingolipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL, USA); caged-S1P was from Enzo Life Sciences Inc. (Farmingdale, NY, USA); human TGF-β2 , TNFα, and IL-1β were from Peprotech (London, UK); BAF312 was from Selleck Chemicals Inc. (Houston, TX, USA); CYM5442, CYM5520, CYM5541, puromycin, the lentiviral particles of human Spns2 and SK-1 shRNA constructs (MISSION® shRNA), diamidine-phenylindole (DAPI), and the antibodies against α-tubulin and Spns2 (SAB2104271) were from Sigma Aldrich Chemikalien, (Buchs, Switzerland); antibodies against phospho-ERK1/2, phospho-Smad2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against CTGF (L-20) and fibronectin (EP5) were from Santa Cruz (Heidelberg, Germany); the AQP1 antibody ( (B)) was from Abcam plc (Cambridge, UK); the human SK-1 antibody was generated and characterized, as previously described [ , ]. .. The secondary anti-rabbit and anti-mouse horseradish peroxidase-coupled IgG antibodies and the enhanced chemiluminescence (ECL) reagents were from GE Health Care Systems GmbH (Freiburg, Germany).

Inhibition:

Article Title: Anorexic response to rapamycin does not appear to involve a central mechanism
Article Snippet: Inhibition of mTOR signalling was assessed by the amount of phosphorylated S6 (pS6) protein in the liver, MBH and HB by Western blot analysis. .. The pS6 antibody (4856S, Cell Signalling, Danvers, MA, USA) and total S6 antibody (2217S, Cell Signalling) were used in 1/1000; while glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (3683S, Cell Signalling) antibody was used in 1/10000 concentrations for normalization.

Imaging:

Article Title: Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation
Article Snippet: Western blot analysis Protein lysates from cells were subjected to SDS-PAGE and target proteins were detected with primary antibodies recognizing p-p38, p38, P-extracellular signal-regulated kinase (P-ERK), ERK, p-JNK, JNK, p-NF-κB p65 (Ser536), NF-κβ p65 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST, Danvers, Massachusetts, USA), PGC-1β and RANKL (Epitomics, San Francisco, USA), MMP-3 and MMP-13 (Abcam, Cambridge, USA) respectively. .. After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (EarthOx, Millbrae, California, USA), protein bands were visualized using enhanced chemiluminescence (Millipore, Boston, USA) plus western blot detection reagents followed by exposure to a scanning imager (G:BOX Gel & Blot Imaging Series from Syngene, Cambridge, UK).

Protein Concentration:

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Protein concentration was quantified using the Pierce BCA Protein Assay kit (Pierce Biotechnology Inc., Rockford, IL, USA). .. Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK).

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats
Article Snippet: The protein concentration was assayed using the Bradford protein method. .. The membranes were blocked in 5% dry milk for 1 h and incubated overnight with one of the following primary antibodies at 4°C: total AKT (1∶1000, Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Ser1177 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Thr495 (1∶500, Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Cell Signaling Technology, Danvers, MA, USA), and Mas (1∶500) .

Polymerase Chain Reaction:

Article Title: IMP1 regulates UCA1-mediated cell invasion through facilitating UCA1 decay and decreasing the sponge effect of UCA1 for miR-122-5p
Article Snippet: Reagents Sources of primary antibodies were IMP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Cell Signaling (Danvers, USA); CNOT1 and Ago2 from Santa Cruz Biotechnology (Dallas, USA); green fluorescent protein (GFP) and Flag from Sigma Aldrich (USA). .. Polymerase chain reaction (PCR) primers used in the study were ordered from Tiangen Biotech Co. (Beijing, China) and are listed in Additional file : Table S1.

Nucleic Acid Electrophoresis:

Article Title: Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy
Article Snippet: .. Proteins from total lysates were resolved by 8 to 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Akt and phospho-Ser473 Akt, p70 S6K and phospho-Thr389 p70 S6K, and eIF2α and phospho-Ser51 eIF2α (1:1000 to 1:2000, Cell Signaling Technology). ..

Isolation:

Article Title: Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B
Article Snippet: Protein concentrations of extracts from mouse tissues were isolated and extracted as previously described., Protein content was determined using BSA as a standard according to the Bradford assay. .. Blots were probed with antibodies against dysferlin (1:2,500, mouse monoclonal antibody), TRIM72/MG53 (1:2,500, custom rabbit polyclonal antibody), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000, D16H11; Cell Signaling), and appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology).

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Myocardial tissue samples isolated at the time of sacrifice were immediately frozen in liquid nitrogen and stored at −80 °C. .. Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK).

Purification:

Article Title: Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces
Article Snippet: .. Antibodies The following mouse and rabbit monoclonal/polyclonal antibodies (Abs) were used: 5H10, mouse Ab against N-terminus of Dsg3 (sc-23912, Santa Cruz, Dallas, TX, USA); H-145, rabbit Ab against C-terminus of Dsg3 (sc-20116, Santa Cruz, Heidelberg, Germany); 33–3D, mouse IgM against Dsg2 (gift from Professor Garrod); rabbit Ab to Dsc2 (610120, Progen, Heidelberg, Germany); Dsc3-U114, mouse Ab to Dsc3 (65193, Progen); PG 5.1, mouse Ab to Plakoglobin (65015, Progen); 115F, mouse Ab to Desmoplakin (gift from Professor Garrod); H-300, rabbit Ab to Desmoplakin (sc-33555, Santa Cruz); 5C2, mouse Ab to plakophilin1 (Progen); PKP3, mouse Ab (ab151401, Abcam, Cambridge, UK), HECD-1, mouse anti-N-terminus of E-cadherin (ab1416, Abcam); 6F9, mouse anti-β-catenin ascites fluid (C7082, Sigma-Aldrich, Dorset, UK); rabbit Ab to α-catenin (ab2981, Abcam); H-90, rabbit Ab to p120 (sc-13957, Santa Cruz); mouse Ab to β-actin (8H10D10, Cell Signaling Technology); mouse Ab to Phospho-Myosin Light Chain 2 (Ser19) (3671S, Cell Signaling Technology, Leiden, Netherlands); mouse Ab to K14 (gift from Professor Leigh); Alexa Fluor 488 conjugated phalloidin for F-actin (A12379, ThermoFisher Scientific); Alexa Flour 488 conjugated rabbit Ab to non-muscle myosin IIa (ab204675, Abcam); D8H1X, rabbit Ab to YAP (D8H1X-XP, Cell Signaling Technology); EP1675Y, rabbit Ab to YAP1 (phosphor S127) (ab76252, Abcam); C-20, rabbit Ab to FOXM1 (sc-502, Santa Cruz); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Y69, rabbit Ab to c-Myc (ab32072, Abcam); 8H10D10, mouse Ab to beta actin (3700S, Cell Signaling Technology); mouse Ab to 14-3-3 gamma (Santa Cruz); normal rabbit IgG (2729S, Cell Signaling Technology); purified mouse IgG1(401401, Biolegend, London, United Kingdom); 14C10, rabbit Ab to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Loading control (14c10, Cell Signaling Technology); B-6, mouse Ab to heat shock cognate 71 kDa protein (Hsc70)-Loading control (sc-7298, Santa Cruz); anti-Lamin A antibody (ab26300, Abcam); Secondary Abs were anti-mouse/rabbit IgG peroxidase antibody produced in goat (A0168/A6667; Sigma-Aldrich); Alexa Fluor 488 goat anti-mouse/rabbit IgG (A11029/A11034; Invitrogen, Dartford, United Kingdom), and Alexa Fluor 568 goat anti-mouse/rabbit IgG (A11031/A11036; ThermoFisher Scientific). .. Cell culture and siRNA Transfection Various epithelial cell types were used in the study; HaCaT immortalized human skin keratinocyte line, SqCC/Y1 human oral buccal squamous cell carcinoma (SCC) line, a cutaneous squamous cell carcinoma T8 cell line (gift from Professor Harwood) with the transduction of empty vector control (Vect Ct) and full length Dsg3 (FL) and primary keratinocytes derived from foreskin and breast skin and oral mucous tissues.

Protein Extraction:

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Tissues were placed in lysing matrix tubes (QBiogene/MP Biomedicals, Solon, OH, USA), mixed with 1 mL of protein extraction buffer containing 10 mM imidazole, 300 mM sucrose, 1 mM dithiotreitol, 1mM sodium metabisulfite, 25 mM sodium fluoride, 5 mM sodium ethylenediaminetetraacetic acid, 5 mM sodium pyrophosphate, 0.3 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche Diagnostics Belgium, Vilvoorde, Belgium) (Lenaerts et al., 2013), and homogenized in the FastPrep24 instrument (MP Biomedicals). .. Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK).

Staining:

Article Title: Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells
Article Snippet: Crystal violet staining solution and DNA loading buffer were obtained from Beyotime Inc. (Haimen, China). .. Specific antibodies against Bcl-2, bcl-X protein (Bcl-XL), bcl-2-associated X protein (BAX), bcl-2 homologous antagonist-killer protein (BAK), poly-ADP-ribose polymerase (PARP), caspase-3, caspase-9, phospho-c-Jun N-terminal protein kinase (p-JNK), phospho-extracellular regulated protein kinase 1/2 (p-ERK), phospho-p38 mitogen-activated protein kinase (p-p38), JNK, p38, ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and related secondary antibodies were purchased from Cell Signaling Technology (Beverly, USA).

Mouse Assay:

Article Title: Targeted therapy for the post-operative conjunctiva: SPARC silencing reduces collagen deposition
Article Snippet: Operated conjunctival tissues from five mice were pooled as one group in a total of three groups for each condition (n=3). .. Antibodies against SPARC, collagen 1A1 (COL1A1), FN1, MMP-14 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signalling Technology (Massachusetts, USA), Abnova Corp. (Colorado, USA), Epitomics (Abcam, Massachusetts, USA), Abcam (Massachusetts, USA) and Santa Cruz Biotechnology (California, USA), respectively.

SDS Page:

Article Title: Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B
Article Snippet: Protein samples (10–20 μg/lane) were separated by SDS-PAGE at room temperature on 4%–15% gradient gels at 120 V and were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) through electroblotting. .. Blots were probed with antibodies against dysferlin (1:2,500, mouse monoclonal antibody), TRIM72/MG53 (1:2,500, custom rabbit polyclonal antibody), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000, D16H11; Cell Signaling), and appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology).

Article Title: Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation
Article Snippet: .. Western blot analysis Protein lysates from cells were subjected to SDS-PAGE and target proteins were detected with primary antibodies recognizing p-p38, p38, P-extracellular signal-regulated kinase (P-ERK), ERK, p-JNK, JNK, p-NF-κB p65 (Ser536), NF-κβ p65 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST, Danvers, Massachusetts, USA), PGC-1β and RANKL (Epitomics, San Francisco, USA), MMP-3 and MMP-13 (Abcam, Cambridge, USA) respectively. .. After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (EarthOx, Millbrae, California, USA), protein bands were visualized using enhanced chemiluminescence (Millipore, Boston, USA) plus western blot detection reagents followed by exposure to a scanning imager (G:BOX Gel & Blot Imaging Series from Syngene, Cambridge, UK).

Plasmid Preparation:

Article Title: Molecular mechanisms involved in HIV-1 Tat-mediated induction of IL-6 and IL-8 in astrocytes
Article Snippet: HIV-1 Tat expression plasmid, initially developed by Dr. E Verdin, Gladstone institute, UCSF (catalog # 10453), and HIV-1 Tat protein (catalog # 2222) were obtained from the NIH AIDS reagent program. .. The primary antibodies for p65, p-c-jun, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and all the secondary antibodies were purchased from Cell Signaling (Danvers, MA, USA) and primary antibodies for p-p38, p-Akt, p-JNK and LaminB were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Software:

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages
Article Snippet: Each membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies against IL-1β , Iκ Bα , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IKKβ (Ser177/181), IKKα /β , phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, NLRP3, caspase-8 (Cell Signaling Technology, Danvers, MA, USA), pro-IL-18 (Medical & Biological Laboratories), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase-1 p20 (AdipoGen, San Diego, CA, USA) at a dilution of 1/1,000. .. The densities of protein bands were quantified using the ImageJ software (available from the National Institutes of Health, Bethesda, MD, USA).

Article Title: Treatment with Recombinant Human MG53 Protein Increases Membrane Integrity in a Mouse Model of Limb Girdle Muscular Dystrophy 2B
Article Snippet: Blots were probed with antibodies against dysferlin (1:2,500, mouse monoclonal antibody), TRIM72/MG53 (1:2,500, custom rabbit polyclonal antibody), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:10,000, D16H11; Cell Signaling), and appropriate horseradish peroxidase (HRP)–conjugated secondary antibodies (1:5,000; Santa Cruz Biotechnology). .. Bio-Rad Image Lab (version 4.1) and ImageJ software were used for blot analysis.

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK). .. Protein expression was detected with Super signal west pico chemilumninescent reagents (Thermo Scientific, Rockford, IL, USA) and quantified using Image lab TM Analyzer software (Bio-Rad laboratories N.V.).

Article Title: Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells
Article Snippet: Antibodies specific for phospho-Src family (Tyr416) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA). .. For signal quantitation, the bands were digitalized and analyzed by ImageJ software.

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats
Article Snippet: The membranes were blocked in 5% dry milk for 1 h and incubated overnight with one of the following primary antibodies at 4°C: total AKT (1∶1000, Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Ser1177 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Thr495 (1∶500, Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Cell Signaling Technology, Danvers, MA, USA), and Mas (1∶500) . .. Protein band detection was performed using the Odyssey scanning system (Li-Cor, USA) using Odyssey software.

Electrophoresis:

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages
Article Snippet: The denatured proteins were separated by electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel and then transferred onto a polyvinylidene difluoride membrane (GE Healthcare, Buckinghamshire, UK). .. Each membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies against IL-1β , Iκ Bα , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IKKβ (Ser177/181), IKKα /β , phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, NLRP3, caspase-8 (Cell Signaling Technology, Danvers, MA, USA), pro-IL-18 (Medical & Biological Laboratories), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase-1 p20 (AdipoGen, San Diego, CA, USA) at a dilution of 1/1,000.

shRNA:

Article Title: Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells
Article Snippet: .. S1P and all C17-sphingolipid standards were from Avanti Polar Lipids Inc. (Alabaster, AL, USA); caged-S1P was from Enzo Life Sciences Inc. (Farmingdale, NY, USA); human TGF-β2 , TNFα, and IL-1β were from Peprotech (London, UK); BAF312 was from Selleck Chemicals Inc. (Houston, TX, USA); CYM5442, CYM5520, CYM5541, puromycin, the lentiviral particles of human Spns2 and SK-1 shRNA constructs (MISSION® shRNA), diamidine-phenylindole (DAPI), and the antibodies against α-tubulin and Spns2 (SAB2104271) were from Sigma Aldrich Chemikalien, (Buchs, Switzerland); antibodies against phospho-ERK1/2, phospho-Smad2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were from Cell Signaling Technology (Danvers, MA, USA); antibodies against CTGF (L-20) and fibronectin (EP5) were from Santa Cruz (Heidelberg, Germany); the AQP1 antibody ( (B)) was from Abcam plc (Cambridge, UK); the human SK-1 antibody was generated and characterized, as previously described [ , ]. .. The secondary anti-rabbit and anti-mouse horseradish peroxidase-coupled IgG antibodies and the enhanced chemiluminescence (ECL) reagents were from GE Health Care Systems GmbH (Freiburg, Germany).

Radio Immunoprecipitation:

Article Title: Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells
Article Snippet: Specific antibodies against Bcl-2, bcl-X protein (Bcl-XL), bcl-2-associated X protein (BAX), bcl-2 homologous antagonist-killer protein (BAK), poly-ADP-ribose polymerase (PARP), caspase-3, caspase-9, phospho-c-Jun N-terminal protein kinase (p-JNK), phospho-extracellular regulated protein kinase 1/2 (p-ERK), phospho-p38 mitogen-activated protein kinase (p-p38), JNK, p38, ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and related secondary antibodies were purchased from Cell Signaling Technology (Beverly, USA). .. Radio immunoprecipitation assay (RIPA) lysis buffer was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, USA).

Article Title: Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy
Article Snippet: Cells were rinsed once in ice-cold PBS and harvested in radioimmunoprecipitation assay (RIPA) lysis buffer containing protease inhibitors (Roche) and a cocktail of phosphatase inhibitors (Sigma). .. Proteins from total lysates were resolved by 8 to 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Akt and phospho-Ser473 Akt, p70 S6K and phospho-Thr389 p70 S6K, and eIF2α and phospho-Ser51 eIF2α (1:1000 to 1:2000, Cell Signaling Technology).

Incubation:

Article Title: Regular Voluntary Exercise Potentiates Interleukin-1β and Interleukin-18 Secretion by Increasing Caspase-1 Expression in Murine Macrophages
Article Snippet: .. Each membrane was blocked with 5% bovine serum albumin (Sigma-Aldrich) and incubated with primary antibodies against IL-1β , Iκ Bα , glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-IKKβ (Ser177/181), IKKα /β , phospho-JNK (Thr183/Thr185), JNK, phospho-p38 (Thr180/Tyr182), p38, NLRP3, caspase-8 (Cell Signaling Technology, Danvers, MA, USA), pro-IL-18 (Medical & Biological Laboratories), ASC (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase-1 p20 (AdipoGen, San Diego, CA, USA) at a dilution of 1/1,000. .. The secondary antibodies horseradish peroxidase-conjugated AffiniPure Mouse Anti-Rabbit IgG or Goat Anti-Mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used at a dilution of 1 : 20,000.

Article Title: Reconstituted HDL (Milano) Treatment Efficaciously Reverses Heart Failure with Preserved Ejection Fraction in Mice
Article Snippet: .. Membranes were incubated with primary antibodies against acetyl-coenzyme A (acetyl-CoA) carboxylase (ACC), p-ACC (Ser79), transforming growth factor (TGF)-β1, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all prior antibodies from Cell Signaling Technologies, Beverly, MA, USA), and osteopontin (Abcam, Cambridge, UK). .. Protein expression was detected with Super signal west pico chemilumninescent reagents (Thermo Scientific, Rockford, IL, USA) and quantified using Image lab TM Analyzer software (Bio-Rad laboratories N.V.).

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats
Article Snippet: .. The membranes were blocked in 5% dry milk for 1 h and incubated overnight with one of the following primary antibodies at 4°C: total AKT (1∶1000, Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Ser1177 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Thr495 (1∶500, Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Cell Signaling Technology, Danvers, MA, USA), and Mas (1∶500) . .. Protein band detection was performed using the Odyssey scanning system (Li-Cor, USA) using Odyssey software.

Article Title: Down-regulating peroxisome proliferator-activated receptor-gamma coactivator-1beta alleviates the proinflammatory effect of rheumatoid arthritis fibroblast-like synoviocytes through inhibiting extracellular signal-regulated kinase, p38 and nuclear factor-kappaB activation
Article Snippet: Western blot analysis Protein lysates from cells were subjected to SDS-PAGE and target proteins were detected with primary antibodies recognizing p-p38, p38, P-extracellular signal-regulated kinase (P-ERK), ERK, p-JNK, JNK, p-NF-κB p65 (Ser536), NF-κβ p65 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (CST, Danvers, Massachusetts, USA), PGC-1β and RANKL (Epitomics, San Francisco, USA), MMP-3 and MMP-13 (Abcam, Cambridge, USA) respectively. .. After incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (EarthOx, Millbrae, California, USA), protein bands were visualized using enhanced chemiluminescence (Millipore, Boston, USA) plus western blot detection reagents followed by exposure to a scanning imager (G:BOX Gel & Blot Imaging Series from Syngene, Cambridge, UK).

Produced:

Article Title: Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces
Article Snippet: .. Antibodies The following mouse and rabbit monoclonal/polyclonal antibodies (Abs) were used: 5H10, mouse Ab against N-terminus of Dsg3 (sc-23912, Santa Cruz, Dallas, TX, USA); H-145, rabbit Ab against C-terminus of Dsg3 (sc-20116, Santa Cruz, Heidelberg, Germany); 33–3D, mouse IgM against Dsg2 (gift from Professor Garrod); rabbit Ab to Dsc2 (610120, Progen, Heidelberg, Germany); Dsc3-U114, mouse Ab to Dsc3 (65193, Progen); PG 5.1, mouse Ab to Plakoglobin (65015, Progen); 115F, mouse Ab to Desmoplakin (gift from Professor Garrod); H-300, rabbit Ab to Desmoplakin (sc-33555, Santa Cruz); 5C2, mouse Ab to plakophilin1 (Progen); PKP3, mouse Ab (ab151401, Abcam, Cambridge, UK), HECD-1, mouse anti-N-terminus of E-cadherin (ab1416, Abcam); 6F9, mouse anti-β-catenin ascites fluid (C7082, Sigma-Aldrich, Dorset, UK); rabbit Ab to α-catenin (ab2981, Abcam); H-90, rabbit Ab to p120 (sc-13957, Santa Cruz); mouse Ab to β-actin (8H10D10, Cell Signaling Technology); mouse Ab to Phospho-Myosin Light Chain 2 (Ser19) (3671S, Cell Signaling Technology, Leiden, Netherlands); mouse Ab to K14 (gift from Professor Leigh); Alexa Fluor 488 conjugated phalloidin for F-actin (A12379, ThermoFisher Scientific); Alexa Flour 488 conjugated rabbit Ab to non-muscle myosin IIa (ab204675, Abcam); D8H1X, rabbit Ab to YAP (D8H1X-XP, Cell Signaling Technology); EP1675Y, rabbit Ab to YAP1 (phosphor S127) (ab76252, Abcam); C-20, rabbit Ab to FOXM1 (sc-502, Santa Cruz); H-432, rabbit Ab to Cyclin A (sc-751, Santa Cruz); PC10, mouse Ab to PCNA (sc-56, Santa Cruz); Y69, rabbit Ab to c-Myc (ab32072, Abcam); 8H10D10, mouse Ab to beta actin (3700S, Cell Signaling Technology); mouse Ab to 14-3-3 gamma (Santa Cruz); normal rabbit IgG (2729S, Cell Signaling Technology); purified mouse IgG1(401401, Biolegend, London, United Kingdom); 14C10, rabbit Ab to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-Loading control (14c10, Cell Signaling Technology); B-6, mouse Ab to heat shock cognate 71 kDa protein (Hsc70)-Loading control (sc-7298, Santa Cruz); anti-Lamin A antibody (ab26300, Abcam); Secondary Abs were anti-mouse/rabbit IgG peroxidase antibody produced in goat (A0168/A6667; Sigma-Aldrich); Alexa Fluor 488 goat anti-mouse/rabbit IgG (A11029/A11034; Invitrogen, Dartford, United Kingdom), and Alexa Fluor 568 goat anti-mouse/rabbit IgG (A11031/A11036; ThermoFisher Scientific). .. Cell culture and siRNA Transfection Various epithelial cell types were used in the study; HaCaT immortalized human skin keratinocyte line, SqCC/Y1 human oral buccal squamous cell carcinoma (SCC) line, a cutaneous squamous cell carcinoma T8 cell line (gift from Professor Harwood) with the transduction of empty vector control (Vect Ct) and full length Dsg3 (FL) and primary keratinocytes derived from foreskin and breast skin and oral mucous tissues.

Immunoprecipitation:

Article Title: Nuclear lymphocyte-specific protein tyrosine kinase and its interaction with CR6-interacting factor 1 promote the survival of human leukemic T cells
Article Snippet: Paragraph title: Immunoprecipitation and immunoblotting ... Antibodies specific for phospho-Src family (Tyr416) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Lysis:

Article Title: Isocryptotanshinone Induced Apoptosis and Activated MAPK Signaling in Human Breast Cancer MCF-7 Cells
Article Snippet: Specific antibodies against Bcl-2, bcl-X protein (Bcl-XL), bcl-2-associated X protein (BAX), bcl-2 homologous antagonist-killer protein (BAK), poly-ADP-ribose polymerase (PARP), caspase-3, caspase-9, phospho-c-Jun N-terminal protein kinase (p-JNK), phospho-extracellular regulated protein kinase 1/2 (p-ERK), phospho-p38 mitogen-activated protein kinase (p-p38), JNK, p38, ERK, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and related secondary antibodies were purchased from Cell Signaling Technology (Beverly, USA). .. Radio immunoprecipitation assay (RIPA) lysis buffer was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, USA).

Article Title: Fasting Cycles Retard Growth of Tumors and Sensitize a Range of Cancer Cell Types to Chemotherapy
Article Snippet: Tumor tissues were homogenized in RIPA lysis buffer supplemented with the same protease and phosphatase inhibitors. .. Proteins from total lysates were resolved by 8 to 12% SDS–polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Akt and phospho-Ser473 Akt, p70 S6K and phospho-Thr389 p70 S6K, and eIF2α and phospho-Ser51 eIF2α (1:1000 to 1:2000, Cell Signaling Technology).

Article Title: The Novel Mas agonist, CGEN-856S, Attenuates Isoproterenol-Induced Cardiac Remodeling and Myocardial Infarction Injury in Rats
Article Snippet: The remaining cells were scraped into 180 µL of lysis buffer (50 mM Na4 P2 O7 , 50 mM NaF, 5 mM Na2 EDTA, 5 mM NaCl, 5 mM EGTA, 10 mM HEPES, 1% Triton X-100, and a specific EDTA-free inhibitor cocktail) for each cell culture flask (75 cm2 ). .. The membranes were blocked in 5% dry milk for 1 h and incubated overnight with one of the following primary antibodies at 4°C: total AKT (1∶1000, Cell Signaling Technology, Danvers, MA, USA), p-AKT Ser473 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Ser1177 (1∶500, Cell Signaling Technology, Danvers, MA, USA), p-eNOS Thr495 (1∶500, Cell Signaling Technology, Danvers, MA, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1∶1000, Cell Signaling Technology, Danvers, MA, USA), and Mas (1∶500) .

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Cell Signaling Technology Inc rabbit anti bax
    Effect of resveratrol involved in degradation of <t>Bcl-2</t> and release of cytochrome c in human melanoma cells. Notes: ( A and B ) shGFP- or shp53-transduced MV3 cells were treated with or without resveratrol (200 μM, 48 hours). A qRT-PCR was performed to examine mRNA levels of Bcl-2 and <t>Bax.</t> All data were shown as the mean ± SD, ** P
    Rabbit Anti Bax, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti bax/product/Cell Signaling Technology Inc
    Average 99 stars, based on 93 article reviews
    Price from $9.99 to $1999.99
    rabbit anti bax - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc gapdh
    Examination of TNF-α-induced NF-κB p65 nuclear translocation and degradation of IκBα in <t>HTNV-infected</t> cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and HTNV N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, <t>GAPDH,</t> or histone H2B.
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 425 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh/product/Cell Signaling Technology Inc
    Average 99 stars, based on 425 article reviews
    Price from $9.99 to $1999.99
    gapdh - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc glyceraldehyde 3 phosphate dehydrogenase gapdh
    P2X7 and EGF Receptor Stimulation Regulates the Expression of DUSP6 in Cerebellar Granule Neurons and Astrocytes ) for different times and DUSP6 and <t>Glyceraldehyde-3-phosphate</t> dehydrogenase <t>(GAPDH)</t> were detected in the total lysates by immunoblotting. (A) Immunoblots of a representative time-course experiment for each effector. (B) Cells were incubated with or without the effectors, 300 μM BzATP or 100 ng/mL EGF, for 30 min, fixed and the presence of DUSP6 was detected by immunocytochemistry. Representative immunofluorescence images are shown. Scale bars represent 50 μm. (C) The diagrams represent the quantification of the time courses obtained by normalization to the corresponding GAPDH levels. The data are represented as the means ± SEM from three independent experiments performed on different cultures. *** p
    Glyceraldehyde 3 Phosphate Dehydrogenase Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glyceraldehyde 3 phosphate dehydrogenase gapdh/product/Cell Signaling Technology Inc
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    glyceraldehyde 3 phosphate dehydrogenase gapdh - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    99
    Cell Signaling Technology Inc glyceraldehyde 3 phosphate dehydrogenase
    Mitochondrial biogenesis related‐protein and gene expressions Quantitative analysis of the phosphorylated form (Thr172) of AMP kinase (AMPK) protein ( A ) and gene expressions of sirtuin1 ( Sirt1 ), peroxisome proliferator‐activated receptor γ coactivator 1 ( Pgc‐1 ), nuclear respiratory factor 1 ( Nrf‐1 ) and mitochondrial transcription factor A ( Tfam ) mRNA ( B ) in skeletal muscle obtained from the ND, ND+Sesamin, HFD and HFD+Sesamin mice ( n  = 6–8 for each group). Protein expression was normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Gene expressions were normalized to GAPDH and depicted as the ratio to ND. Data are shown as means + SEM. * P
    Glyceraldehyde 3 Phosphate Dehydrogenase, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glyceraldehyde 3 phosphate dehydrogenase/product/Cell Signaling Technology Inc
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glyceraldehyde 3 phosphate dehydrogenase - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

    Image Search Results


    Effect of resveratrol involved in degradation of Bcl-2 and release of cytochrome c in human melanoma cells. Notes: ( A and B ) shGFP- or shp53-transduced MV3 cells were treated with or without resveratrol (200 μM, 48 hours). A qRT-PCR was performed to examine mRNA levels of Bcl-2 and Bax. All data were shown as the mean ± SD, ** P

    Journal: OncoTargets and therapy

    Article Title: Resveratrol induces apoptosis in human melanoma cell through negatively regulating Erk/PKM2/Bcl-2 axis

    doi: 10.2147/OTT.S186247

    Figure Lengend Snippet: Effect of resveratrol involved in degradation of Bcl-2 and release of cytochrome c in human melanoma cells. Notes: ( A and B ) shGFP- or shp53-transduced MV3 cells were treated with or without resveratrol (200 μM, 48 hours). A qRT-PCR was performed to examine mRNA levels of Bcl-2 and Bax. All data were shown as the mean ± SD, ** P

    Article Snippet: Antibodies and reagents The following antibodies and reagents were used for this study: rabbit anti-p53 (1:500, ab1431; Abcam), rabbit anti-p53 (acetyl K382) (1:1,000, ab75754; Abcam), rabbit anti-p21 (1:500, ab109520; Abcam), rabbit anti-gamma H2AX (phospho S139) (1:1,000, ab2893; Abcam), rabbit anti-ERK1/2 (1:500, No 5013; Cell Signaling Technology Inc., Danvers, MA, USA), rabbit anti-phospho-ERK1/2 (Thr202/Tyr204) (1:2,000, No 4376; Cell Signaling Technology Inc.), rabbit anti-PKM2 (1:1,000, ab150377; Abcam), rabbit anti-β-actin (1:2,000, ab16039; Abcam), mouse anti-Tubulin (1:2,000, AT819; Beyotime Institute of Biotechnology), rabbit anti-active caspase3 (1:1,000, ab49822; Abcam), mouse anti-cleaved PARP1 (1:1,000, ab198490; Abcam), mouse anti-cytochrome C (1:2,000, ab13575; Abcam), rabbit anti-VDAC (1:1,000, ab15895; Abcam), mouse anti-Bcl-2 (1:1,000, ab201566; Abcam), rabbit anti-Bax (1:500, No 2774; Cell Signaling Technology Inc.), and mouse-anti-HA (1:500, H3663; Sigma-Aldrich Co.).

    Techniques: Quantitative RT-PCR

    Examination of TNF-α-induced NF-κB p65 nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and HTNV N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Examination of TNF-α-induced NF-κB p65 nuclear translocation and degradation of IκBα in HTNV-infected cells. A549 cells were mock infected (M) or infected with HTNV (V) at an MOI of 5. On day 5, mock- and HTNV-infected cells were left untreated (−) or treated (+) with 50 ng/ml of TNF-α for 15 min. (A) After fixation, cells were stained with antibodies against NF-κB p65 (green) and HTNV N protein (red) and stained with DAPI to highlight nuclei (blue). (B) Cell lysates were separated into cytoplasmic and nuclear fractions. (C) Total cell lysates were prepared for immunoblotting, and proteins were transferred onto PVDF membranes. Blots were probed with antibodies against IκBα, p50, p65, HTNV N protein, GAPDH, or histone H2B.

    Article Snippet: Blots were blocked with 5% nonfat milk in Tris-buffered saline (TBS) and subsequently probed overnight at 4°C with antibodies directed against p50 (Santa Cruz Biotechnologies), p65 (Santa Cruz Biotechnologies), HTNV N (rabbit immune sera), IκBα (Santa Cruz Biotechnologies), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Cell Signaling Technology), histone H2B (Santa Cruz Biotechnologies), and FLAG (M2; Sigma-Aldrich).

    Techniques: Translocation Assay, Infection, Staining

    Endogenous NF-κB transcription activation in cells expressing HTNV N protein. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After treatment of the cells with 50 ng/ml of TNF-α for 15 min, cytoplasmic and nuclear extracts from lysates were prepared. (A) Nuclear extracts were allowed to bind to NF-κB consensus sequence oligonucleotides on 96-well plates and then probed with antibodies specific for NF-κB p65 or NF-κB p50. The absorbance reading for each sample was determined using a spectrophotometer. OD, optical density; MUT oligo, mutated consensus oligonucleotide; WT oligo, wild-type consensus oligonucleotide. (B) Cytoplasmic extracts were used for immunoblotting to detect HTNV N protein and GAPDH. Each point represents an average ± standard deviation of results for six samples. The statistical significance of results for HTNV S with TNF and MG132 with TNF was determined by comparing the results to those for the empty vector with TNF. Asterisks indicate significant differences ( P

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Endogenous NF-κB transcription activation in cells expressing HTNV N protein. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. After treatment of the cells with 50 ng/ml of TNF-α for 15 min, cytoplasmic and nuclear extracts from lysates were prepared. (A) Nuclear extracts were allowed to bind to NF-κB consensus sequence oligonucleotides on 96-well plates and then probed with antibodies specific for NF-κB p65 or NF-κB p50. The absorbance reading for each sample was determined using a spectrophotometer. OD, optical density; MUT oligo, mutated consensus oligonucleotide; WT oligo, wild-type consensus oligonucleotide. (B) Cytoplasmic extracts were used for immunoblotting to detect HTNV N protein and GAPDH. Each point represents an average ± standard deviation of results for six samples. The statistical significance of results for HTNV S with TNF and MG132 with TNF was determined by comparing the results to those for the empty vector with TNF. Asterisks indicate significant differences ( P

    Article Snippet: Blots were blocked with 5% nonfat milk in Tris-buffered saline (TBS) and subsequently probed overnight at 4°C with antibodies directed against p50 (Santa Cruz Biotechnologies), p65 (Santa Cruz Biotechnologies), HTNV N (rabbit immune sera), IκBα (Santa Cruz Biotechnologies), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Cell Signaling Technology), histone H2B (Santa Cruz Biotechnologies), and FLAG (M2; Sigma-Aldrich).

    Techniques: Activation Assay, Expressing, Transfection, Sequencing, Spectrophotometry, Standard Deviation, Plasmid Preparation

    Examination of NF-κB p50 and p65 levels and TNF-α-induced IκBα degradation in HTNV N-expressing cells. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. To induce the degradation of IκBα, cells were treated with 50 ng/ml of TNF-α for 15 min, and lysates were prepared for immunoblotting. For uninduced samples, cells were left untreated. Proteins were transferred onto PVDF membranes and probed with antibodies against IκBα, p50, p65, HTNV N protein, or GAPDH. +, with; −, without.

    Journal: Journal of Virology

    Article Title: Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿

    doi: 10.1128/JVI.00986-08

    Figure Lengend Snippet: Examination of NF-κB p50 and p65 levels and TNF-α-induced IκBα degradation in HTNV N-expressing cells. A549 cells were transfected with 500 ng of pWRG7077-Empty, pWRG7077-HTNV-S, or pWRG7077-HTNV-M for 24 h. To induce the degradation of IκBα, cells were treated with 50 ng/ml of TNF-α for 15 min, and lysates were prepared for immunoblotting. For uninduced samples, cells were left untreated. Proteins were transferred onto PVDF membranes and probed with antibodies against IκBα, p50, p65, HTNV N protein, or GAPDH. +, with; −, without.

    Article Snippet: Blots were blocked with 5% nonfat milk in Tris-buffered saline (TBS) and subsequently probed overnight at 4°C with antibodies directed against p50 (Santa Cruz Biotechnologies), p65 (Santa Cruz Biotechnologies), HTNV N (rabbit immune sera), IκBα (Santa Cruz Biotechnologies), GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Cell Signaling Technology), histone H2B (Santa Cruz Biotechnologies), and FLAG (M2; Sigma-Aldrich).

    Techniques: Expressing, Transfection

    P2X7 and EGF Receptor Stimulation Regulates the Expression of DUSP6 in Cerebellar Granule Neurons and Astrocytes ) for different times and DUSP6 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the total lysates by immunoblotting. (A) Immunoblots of a representative time-course experiment for each effector. (B) Cells were incubated with or without the effectors, 300 μM BzATP or 100 ng/mL EGF, for 30 min, fixed and the presence of DUSP6 was detected by immunocytochemistry. Representative immunofluorescence images are shown. Scale bars represent 50 μm. (C) The diagrams represent the quantification of the time courses obtained by normalization to the corresponding GAPDH levels. The data are represented as the means ± SEM from three independent experiments performed on different cultures. *** p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: P2X7 Nucleotide and EGF Receptors Exert Dual Modulation of the Dual-Specificity Phosphatase 6 (MKP-3) in Granule Neurons and Astrocytes, Contributing to Negative Feedback on ERK Signaling

    doi: 10.3389/fnmol.2017.00448

    Figure Lengend Snippet: P2X7 and EGF Receptor Stimulation Regulates the Expression of DUSP6 in Cerebellar Granule Neurons and Astrocytes ) for different times and DUSP6 and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were detected in the total lysates by immunoblotting. (A) Immunoblots of a representative time-course experiment for each effector. (B) Cells were incubated with or without the effectors, 300 μM BzATP or 100 ng/mL EGF, for 30 min, fixed and the presence of DUSP6 was detected by immunocytochemistry. Representative immunofluorescence images are shown. Scale bars represent 50 μm. (C) The diagrams represent the quantification of the time courses obtained by normalization to the corresponding GAPDH levels. The data are represented as the means ± SEM from three independent experiments performed on different cultures. *** p

    Article Snippet: Antisera for Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was generated in rabbit and purchased from Cell Signaling Technology (Beverly, MA, USA).

    Techniques: Expressing, Western Blot, Incubation, Immunocytochemistry, Immunofluorescence

    Mitochondrial biogenesis related‐protein and gene expressions Quantitative analysis of the phosphorylated form (Thr172) of AMP kinase (AMPK) protein ( A ) and gene expressions of sirtuin1 ( Sirt1 ), peroxisome proliferator‐activated receptor γ coactivator 1 ( Pgc‐1 ), nuclear respiratory factor 1 ( Nrf‐1 ) and mitochondrial transcription factor A ( Tfam ) mRNA ( B ) in skeletal muscle obtained from the ND, ND+Sesamin, HFD and HFD+Sesamin mice ( n  = 6–8 for each group). Protein expression was normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Gene expressions were normalized to GAPDH and depicted as the ratio to ND. Data are shown as means + SEM. * P

    Journal: Experimental Physiology

    Article Title: Sesamin prevents decline in exercise capacity and impairment of skeletal muscle mitochondrial function in mice with high‐fat diet‐induced diabetes

    doi: 10.1113/EP085251

    Figure Lengend Snippet: Mitochondrial biogenesis related‐protein and gene expressions Quantitative analysis of the phosphorylated form (Thr172) of AMP kinase (AMPK) protein ( A ) and gene expressions of sirtuin1 ( Sirt1 ), peroxisome proliferator‐activated receptor γ coactivator 1 ( Pgc‐1 ), nuclear respiratory factor 1 ( Nrf‐1 ) and mitochondrial transcription factor A ( Tfam ) mRNA ( B ) in skeletal muscle obtained from the ND, ND+Sesamin, HFD and HFD+Sesamin mice ( n  = 6–8 for each group). Protein expression was normalized to glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Gene expressions were normalized to GAPDH and depicted as the ratio to ND. Data are shown as means + SEM. * P

    Article Snippet: Equal loading of protein was verified by immunoblotting with glyceraldehyde 3‐phosphate dehydrogenase (GAPDH; Cell Signaling), as previously described (Takada et al .

    Techniques: Pyrolysis Gas Chromatography, Mouse Assay, Expressing