Structured Review

Cell Signaling Technology Inc gapdh
GluN2A and GluN2B protein levels in adult human temporal cortical tissue homogenate show an age-dependent decline. (A) Representative examples of GluN2A blot showing GluN2A and GluN2A-S bands. Quantification for GluN2A (B) and GluN2A-S (C), and GluN2B (B, from blots shown in Fig. 1E ) relative to the constitutive protein <t>GAPDH.</t> Spearman’s coefficient of correlation (r) and p-value (p) are indicated for each measure. (E) Representative blots of GluN2A and GluN2B subunits co-immunoprecipitated with <t>PSD-95</t> using human temporal cortical tissue lysates. Symbols in B, C and D correspond to the neurological condition necessitating neurosurgery as depicted in Figure 1 .
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Images

1) Product Images from "Age dependent changes in synaptic NMDA receptor composition in adult human cortical neurons"

Article Title: Age dependent changes in synaptic NMDA receptor composition in adult human cortical neurons

Journal: bioRxiv

doi: 10.1101/2020.01.21.913475

GluN2A and GluN2B protein levels in adult human temporal cortical tissue homogenate show an age-dependent decline. (A) Representative examples of GluN2A blot showing GluN2A and GluN2A-S bands. Quantification for GluN2A (B) and GluN2A-S (C), and GluN2B (B, from blots shown in Fig. 1E ) relative to the constitutive protein GAPDH. Spearman’s coefficient of correlation (r) and p-value (p) are indicated for each measure. (E) Representative blots of GluN2A and GluN2B subunits co-immunoprecipitated with PSD-95 using human temporal cortical tissue lysates. Symbols in B, C and D correspond to the neurological condition necessitating neurosurgery as depicted in Figure 1 .
Figure Legend Snippet: GluN2A and GluN2B protein levels in adult human temporal cortical tissue homogenate show an age-dependent decline. (A) Representative examples of GluN2A blot showing GluN2A and GluN2A-S bands. Quantification for GluN2A (B) and GluN2A-S (C), and GluN2B (B, from blots shown in Fig. 1E ) relative to the constitutive protein GAPDH. Spearman’s coefficient of correlation (r) and p-value (p) are indicated for each measure. (E) Representative blots of GluN2A and GluN2B subunits co-immunoprecipitated with PSD-95 using human temporal cortical tissue lysates. Symbols in B, C and D correspond to the neurological condition necessitating neurosurgery as depicted in Figure 1 .

Techniques Used: Immunoprecipitation

2) Product Images from "Increased mTOR activity and metabolic efficiency in mouse and human cells containing the African-centric tumor-predisposing p53 variant Pro47Ser"

Article Title: Increased mTOR activity and metabolic efficiency in mouse and human cells containing the African-centric tumor-predisposing p53 variant Pro47Ser

Journal: bioRxiv

doi: 10.1101/2020.02.14.946269

Increased mTOR-Rheb binding in S47 cells, along with altered GAPDH conformation. (A-B) An in situ proximity ligation assay (PLA) was performed in WT and S47 MEFs. Each red dot represents an interaction between mTOR-Rheb or GAPDH-Rheb as indicated, scale bar represents 50 µM. The samples were counterstained with DAPI to detect nuclei. Cells stained only with secondary antibody were used as a negative control. (B) Quantification of the mTOR-Rheb interactions, measured as the average number of PLA signals per nuclei. Data were quantified by counting the number of cells in five random fields per experimental condition. (***) p-value
Figure Legend Snippet: Increased mTOR-Rheb binding in S47 cells, along with altered GAPDH conformation. (A-B) An in situ proximity ligation assay (PLA) was performed in WT and S47 MEFs. Each red dot represents an interaction between mTOR-Rheb or GAPDH-Rheb as indicated, scale bar represents 50 µM. The samples were counterstained with DAPI to detect nuclei. Cells stained only with secondary antibody were used as a negative control. (B) Quantification of the mTOR-Rheb interactions, measured as the average number of PLA signals per nuclei. Data were quantified by counting the number of cells in five random fields per experimental condition. (***) p-value

Techniques Used: Binding Assay, In Situ, Proximity Ligation Assay, Staining, Negative Control

Impact of glutathione depletion in S47 cells. (A) WT and S47 MEFs were untreated or treated with 50 µM of DEM for 5 h. Cell lysates were subjected to Western blot analysis and immunoblotted for total mTOR, Rheb and GAPDH. (B) An in situ proximity ligation assay was performed in WT and S47 MEFs that are treated with 10 µM of BSO for 24 h. Shown on the right is the quantification of the mTOR-Rheb interactions, measured as the average number of PLA signals per nuclei. (C) Cell lysates were extracted from WT MEFs treated with 50 µM of DEM for 5 h. Cell lysates were subjected to Western blot analysis and immunoblotted for the proteins indicated. (D) Cell lysates were extracted from two sets of WT and S47 MEFs and were analyzed by Western blot for TSC2, DEPTOR, AKT, p53, Sco2 and GAPDH.
Figure Legend Snippet: Impact of glutathione depletion in S47 cells. (A) WT and S47 MEFs were untreated or treated with 50 µM of DEM for 5 h. Cell lysates were subjected to Western blot analysis and immunoblotted for total mTOR, Rheb and GAPDH. (B) An in situ proximity ligation assay was performed in WT and S47 MEFs that are treated with 10 µM of BSO for 24 h. Shown on the right is the quantification of the mTOR-Rheb interactions, measured as the average number of PLA signals per nuclei. (C) Cell lysates were extracted from WT MEFs treated with 50 µM of DEM for 5 h. Cell lysates were subjected to Western blot analysis and immunoblotted for the proteins indicated. (D) Cell lysates were extracted from two sets of WT and S47 MEFs and were analyzed by Western blot for TSC2, DEPTOR, AKT, p53, Sco2 and GAPDH.

Techniques Used: Western Blot, In Situ, Proximity Ligation Assay

3) Product Images from "Mitochondrial CaMKII causes metabolic reprogramming, energetic insufficiency, and dilated cardiomyopathy"

Article Title: Mitochondrial CaMKII causes metabolic reprogramming, energetic insufficiency, and dilated cardiomyopathy

Journal: bioRxiv

doi: 10.1101/2020.02.13.947564

mtCaMKII hearts show decreased complex I and increased complex II activity and expression (A) Western blot of heart lysates from WT (n=5) and mtCaMKII (n=5) using OxPhos antibody (B) protein expression for complexes I-V normalized to GAPDH. (C) Blue Native gel with mitochondria from WT and mtCaMKII hearts stained with Coomassie blue and (D) in-gel activity assays for complex I. (E and F) Mitochondrial respiration measured under state 2 (substrate alone) and state 3 (substrate plus ADP) conditions in isolated mitochondria from WT (n=7) and mtCaMKII (n=9) hearts. Substrates favored complex I (pyruvate/malate, E) or complex II (succinate/rotenone, F) activity. (G) Western blot with OxPhos antibody and (H) summary data for complex I normalized to CoxIV in heart lysates from WT (n=4), CKmito (n=4), mtCaMKII (n=4) and mtCaMKII x CKmito (n=3) hearts. (I) Complex I activity measured in mitochondria isolated from WT (n=8), CKmito (n=8), mtCaMKII (n=9) and mtCaMKII x CKmito (n=6) hearts. (Data are represented as mean ± SEM, significance was determined using a two-tailed t test or 1 way AVOVA with Tukey’s multiple comparison’s test, ***P
Figure Legend Snippet: mtCaMKII hearts show decreased complex I and increased complex II activity and expression (A) Western blot of heart lysates from WT (n=5) and mtCaMKII (n=5) using OxPhos antibody (B) protein expression for complexes I-V normalized to GAPDH. (C) Blue Native gel with mitochondria from WT and mtCaMKII hearts stained with Coomassie blue and (D) in-gel activity assays for complex I. (E and F) Mitochondrial respiration measured under state 2 (substrate alone) and state 3 (substrate plus ADP) conditions in isolated mitochondria from WT (n=7) and mtCaMKII (n=9) hearts. Substrates favored complex I (pyruvate/malate, E) or complex II (succinate/rotenone, F) activity. (G) Western blot with OxPhos antibody and (H) summary data for complex I normalized to CoxIV in heart lysates from WT (n=4), CKmito (n=4), mtCaMKII (n=4) and mtCaMKII x CKmito (n=3) hearts. (I) Complex I activity measured in mitochondria isolated from WT (n=8), CKmito (n=8), mtCaMKII (n=9) and mtCaMKII x CKmito (n=6) hearts. (Data are represented as mean ± SEM, significance was determined using a two-tailed t test or 1 way AVOVA with Tukey’s multiple comparison’s test, ***P

Techniques Used: Activity Assay, Expressing, Western Blot, Staining, Isolation, Two Tailed Test

Dilated cardiomyopathy in mtCaMKII mice is independent of extramitochondrial CaMKII, MCU phosphorylation, mitochondrial Ca 2+ , ROS, and ΔΨ mito (A) Western blot for flag-CaMKII, VDAC1 and GAPDH in cytoplasm and mitochondria fractions from WT (n=4) and mtCaMKII hearts (n=4) (B) Quantification AC3-I-GFP fusion protein expression in cytoplasm and mitochondria from WT and AC3-I mouse hearts (n=3). (C) Summary data for echocardiographic measurements from WT (n=16), AC3-I (n=13), mtCaMKII (n=18), and mtCaMKII x AC3-I (n=12) mice. (D) Summarized echocardiographic measurement data from WT (n=12), mtCaMKIIN (n=8), mtCaMKII (n=10), and mtCaMKII x mtCaMKIIN (n=10) mice. (E) Western blot and (F) summary data for flag-CaMKII normalized to GAPDH in mtCaMKII (n=4) and mtCaMKII x mtCaMKIIN (n=4) hearts. (G) Western blot and (H) summary data for phosphorylated MCU normalized to total MCU for WT (n=4) and mtCaMKII (n=4) mitochondria. (I) Mitochondrial Ca 2+ uptake assay with cell membrane permeabilized adult ventricular myocytes from WT (n=3) and mtCaMKII (n=3) mice; arrows indicate addition of Ca 2+ . (J) Rate of Ca 2+ uptake calculated from the first 3 peaks of Ca 2+ addition and total Ca 2+ uptake before mPTP opening. (K) Total mitochondrial Ca 2+ content normalized to total protein in isolated mitochondria from WT (n=10) and mtCaMKII (n=10) hearts. (L) H2O2 production measured by Amplex Red in isolated mitochondria from WT (n=8) and mtCaMKII (n=8) hearts. (M) Quantification of TUNEL positive nuclei from heart sections of WT (5 sections from n=4 hearts) and mtCaMKII (5 sections from n=3 hearts) mice. (N) Quantification of fibrosis by Masson’s Trichrome staining from WT (6 sections from n=3 hearts) and mtCaMKII (6 sections from n=3 hearts) heart sections. (O) Mitochondrial membrane potential (ΔΨ mito ) measured with TMRM under state 2 (substrate alone) and state 3 (substrate plus ADP) respiration in isolated mitochondria from WT (n=4) and mtCaMKII (n=4) hearts. (Data are represented as mean ± SEM, significance was determined using a two-tailed t test or 1 way AVOVA with Tukey’s multiple comparison’s test, ****P
Figure Legend Snippet: Dilated cardiomyopathy in mtCaMKII mice is independent of extramitochondrial CaMKII, MCU phosphorylation, mitochondrial Ca 2+ , ROS, and ΔΨ mito (A) Western blot for flag-CaMKII, VDAC1 and GAPDH in cytoplasm and mitochondria fractions from WT (n=4) and mtCaMKII hearts (n=4) (B) Quantification AC3-I-GFP fusion protein expression in cytoplasm and mitochondria from WT and AC3-I mouse hearts (n=3). (C) Summary data for echocardiographic measurements from WT (n=16), AC3-I (n=13), mtCaMKII (n=18), and mtCaMKII x AC3-I (n=12) mice. (D) Summarized echocardiographic measurement data from WT (n=12), mtCaMKIIN (n=8), mtCaMKII (n=10), and mtCaMKII x mtCaMKIIN (n=10) mice. (E) Western blot and (F) summary data for flag-CaMKII normalized to GAPDH in mtCaMKII (n=4) and mtCaMKII x mtCaMKIIN (n=4) hearts. (G) Western blot and (H) summary data for phosphorylated MCU normalized to total MCU for WT (n=4) and mtCaMKII (n=4) mitochondria. (I) Mitochondrial Ca 2+ uptake assay with cell membrane permeabilized adult ventricular myocytes from WT (n=3) and mtCaMKII (n=3) mice; arrows indicate addition of Ca 2+ . (J) Rate of Ca 2+ uptake calculated from the first 3 peaks of Ca 2+ addition and total Ca 2+ uptake before mPTP opening. (K) Total mitochondrial Ca 2+ content normalized to total protein in isolated mitochondria from WT (n=10) and mtCaMKII (n=10) hearts. (L) H2O2 production measured by Amplex Red in isolated mitochondria from WT (n=8) and mtCaMKII (n=8) hearts. (M) Quantification of TUNEL positive nuclei from heart sections of WT (5 sections from n=4 hearts) and mtCaMKII (5 sections from n=3 hearts) mice. (N) Quantification of fibrosis by Masson’s Trichrome staining from WT (6 sections from n=3 hearts) and mtCaMKII (6 sections from n=3 hearts) heart sections. (O) Mitochondrial membrane potential (ΔΨ mito ) measured with TMRM under state 2 (substrate alone) and state 3 (substrate plus ADP) respiration in isolated mitochondria from WT (n=4) and mtCaMKII (n=4) hearts. (Data are represented as mean ± SEM, significance was determined using a two-tailed t test or 1 way AVOVA with Tukey’s multiple comparison’s test, ****P

Techniques Used: Mouse Assay, Western Blot, Expressing, Isolation, TUNEL Assay, Staining, Two Tailed Test

MI data (A) Western blot of cytosolic lysates and summary data for phosphorylated CaMKII and total CaMKII normalized to coomassie staining for sham (n=7) and MI (n=8) mitochondria. Blots for cellular compartment markers also included: SERCA2a (SR membrane), PDH (mitochondrial matrix), GAPDH (cytosol). (B) Western blot for HA-CaMKIIN, VDAC1 and GAPDH in cytoplasm and mitochondria fractions from WT (n=2) and mtCaMKIIN hearts (n=2). (C) Representative images and quantification of of TTC staining in WT and mtCaMKIIN hearts 24 hours after MI surgery. (D) Representative western blot and summary data of pMCU and MCU normalized to coomassie in mitochondrial lysates from WT (n=7 and mtCaMKIIN (n=9) 1 week after MI. (E) qPCR quantification of Ccl2 and Ccl3 mRNA normalized to Hprt in WT (n=7) and mtCaMKIIN (n=11) hearts 1 week after MI. (F) Representative immunofluorescence images and summary data for CD45+ cells in heart sections of WT (n=5 hearts) and mtCaMKIIN (n=5 hearts) hearts 1 week after MI. (Data are represented as mean α SEM, significance was determined using two-tailed t test, (*P
Figure Legend Snippet: MI data (A) Western blot of cytosolic lysates and summary data for phosphorylated CaMKII and total CaMKII normalized to coomassie staining for sham (n=7) and MI (n=8) mitochondria. Blots for cellular compartment markers also included: SERCA2a (SR membrane), PDH (mitochondrial matrix), GAPDH (cytosol). (B) Western blot for HA-CaMKIIN, VDAC1 and GAPDH in cytoplasm and mitochondria fractions from WT (n=2) and mtCaMKIIN hearts (n=2). (C) Representative images and quantification of of TTC staining in WT and mtCaMKIIN hearts 24 hours after MI surgery. (D) Representative western blot and summary data of pMCU and MCU normalized to coomassie in mitochondrial lysates from WT (n=7 and mtCaMKIIN (n=9) 1 week after MI. (E) qPCR quantification of Ccl2 and Ccl3 mRNA normalized to Hprt in WT (n=7) and mtCaMKIIN (n=11) hearts 1 week after MI. (F) Representative immunofluorescence images and summary data for CD45+ cells in heart sections of WT (n=5 hearts) and mtCaMKIIN (n=5 hearts) hearts 1 week after MI. (Data are represented as mean α SEM, significance was determined using two-tailed t test, (*P

Techniques Used: Western Blot, Staining, Real-time Polymerase Chain Reaction, Immunofluorescence, Two Tailed Test

Mitochondrial CaMKII is activated by myocardial infarction and causes dilated cardiomyopathy (A) Western blot of mitochondrial lysates and (B) summary data for phosphorylated CaMKII and total CaMKII and phosphorylated MCU and total MCU normalized to coomassie staining for sham (n=7) and MI (n=8) mitochondria. Blots for cellular compartment markers also included: SERCA2a (SR membrane), PDH (mitochondrial matrix), GAPDH (cytosol). (C) Summary data from echocardiographic measurements from WT (n=6-12) and mtCaMKIIN (n=7-16) mice before, 1 and 3 weeks after MI. EF (left ventricular ejection fraction), EDV (left ventricular end diastolic volume), ESV (left ventricular end systolic volume) (D) Schematic of the mtCaMKII transgene construct, α-MHC: α-myosin heavy chain, cox8a: cox8a mitochondrial localization sequence, Flag: flag epitope tag, HGH1 polyA: human growth hormone polyA signal. (E) Immunofluorescence micrographs of isolated ventricular myocytes from WT and mtCaMKII mice (F) Representative echocardiographic images in end diastole, LV: left ventricle, Ao: aorta (G) Summary data for echocardiographic measurements from adult WT (n=13-20) and mtCaMKII (n=13-23) mice. LVWT (left ventricular wall thickness), HW/BW (heart weight/body weight) (H) Summary data for echocardiographic measurements from young (1-6 weeks of age) WT (n=12) and mtCaMKII (n=6) mice. (I) Representative transmission electron micrographs from WT and mtCaMKII left ventricle. (J) Mitochondrial injury score for WT (n=6) and mtCaMKII (n=6) hearts. (K) Mitochondrial DNA content (cox1) normalized to nuclear DNA (βglobin) for WT (n=4) and mtCaMKII (n=4) hearts. (L) Kaplan-Meier survival relationship for WT (n=41) and mtCaMKII (n=48) mice. (Data are represented as mean ± SEM, significance was determined using a two-tailed t test or log-rank test (survivorship), ****P
Figure Legend Snippet: Mitochondrial CaMKII is activated by myocardial infarction and causes dilated cardiomyopathy (A) Western blot of mitochondrial lysates and (B) summary data for phosphorylated CaMKII and total CaMKII and phosphorylated MCU and total MCU normalized to coomassie staining for sham (n=7) and MI (n=8) mitochondria. Blots for cellular compartment markers also included: SERCA2a (SR membrane), PDH (mitochondrial matrix), GAPDH (cytosol). (C) Summary data from echocardiographic measurements from WT (n=6-12) and mtCaMKIIN (n=7-16) mice before, 1 and 3 weeks after MI. EF (left ventricular ejection fraction), EDV (left ventricular end diastolic volume), ESV (left ventricular end systolic volume) (D) Schematic of the mtCaMKII transgene construct, α-MHC: α-myosin heavy chain, cox8a: cox8a mitochondrial localization sequence, Flag: flag epitope tag, HGH1 polyA: human growth hormone polyA signal. (E) Immunofluorescence micrographs of isolated ventricular myocytes from WT and mtCaMKII mice (F) Representative echocardiographic images in end diastole, LV: left ventricle, Ao: aorta (G) Summary data for echocardiographic measurements from adult WT (n=13-20) and mtCaMKII (n=13-23) mice. LVWT (left ventricular wall thickness), HW/BW (heart weight/body weight) (H) Summary data for echocardiographic measurements from young (1-6 weeks of age) WT (n=12) and mtCaMKII (n=6) mice. (I) Representative transmission electron micrographs from WT and mtCaMKII left ventricle. (J) Mitochondrial injury score for WT (n=6) and mtCaMKII (n=6) hearts. (K) Mitochondrial DNA content (cox1) normalized to nuclear DNA (βglobin) for WT (n=4) and mtCaMKII (n=4) hearts. (L) Kaplan-Meier survival relationship for WT (n=41) and mtCaMKII (n=48) mice. (Data are represented as mean ± SEM, significance was determined using a two-tailed t test or log-rank test (survivorship), ****P

Techniques Used: Western Blot, Staining, Mouse Assay, Construct, Sequencing, FLAG-tag, Immunofluorescence, Isolation, Transmission Assay, Two Tailed Test

4) Product Images from "Acute Endurance Exercise Induces Nuclear p53 Abundance in Human Skeletal Muscle"

Article Title: Acute Endurance Exercise Induces Nuclear p53 Abundance in Human Skeletal Muscle

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2016.00144

Enrichment and purity of protein fractions . Representative Western Blots showing that (A) COXIV is highly abundant in the mitochondrial fraction (A) , and H2B (B) , and GAPDH (C) were enriched in nuclear and cytoplasmic fractions, respectively. M, Mitochondria; N, Nuclear; C, Cytoplasmic.
Figure Legend Snippet: Enrichment and purity of protein fractions . Representative Western Blots showing that (A) COXIV is highly abundant in the mitochondrial fraction (A) , and H2B (B) , and GAPDH (C) were enriched in nuclear and cytoplasmic fractions, respectively. M, Mitochondria; N, Nuclear; C, Cytoplasmic.

Techniques Used: Western Blot

5) Product Images from "Transgender women on oral HIV pre‐exposure prophylaxis have significantly lower tenofovir and emtricitabine concentrations when also taking oestrogen when compared to cisgender men. Transgender women on oral HIV pre‐exposure prophylaxis have significantly lower tenofovir and emtricitabine concentrations when also taking oestrogen when compared to cisgender men"

Article Title: Transgender women on oral HIV pre‐exposure prophylaxis have significantly lower tenofovir and emtricitabine concentrations when also taking oestrogen when compared to cisgender men. Transgender women on oral HIV pre‐exposure prophylaxis have significantly lower tenofovir and emtricitabine concentrations when also taking oestrogen when compared to cisgender men

Journal: Journal of the International AIDS Society

doi: 10.1002/jia2.25405

Kinase expression analysed via immunoblotting for tenofovir‐activating kinases in peripheral blood mononuclear cells (PBMC) and colon tissue. PBMC and colon tissue were lysed and immunoblotting was performed using 50 µg of total protein lysate as described under Methods. (a) , Immunoblotting of PBMC lysate for pyruvate kinase muscle (PKM), pyruvate kinase liver and red blood cell (PKLR), adenylate kinase 2 (AK2) and glyceraldehyde 3‐phosphatase dehydrogenase (GAPDH). (b) , Immunoblotting of colon tissue lysate for creatine kinase muscle (CKM), adenylate kinase 2 (AK2) and β‐actin. Participant identification numbers are indicated (1001, 1002, 1008, 1012, 1017 and 1020). CGM, cisgender male; TGW, transgender woman.
Figure Legend Snippet: Kinase expression analysed via immunoblotting for tenofovir‐activating kinases in peripheral blood mononuclear cells (PBMC) and colon tissue. PBMC and colon tissue were lysed and immunoblotting was performed using 50 µg of total protein lysate as described under Methods. (a) , Immunoblotting of PBMC lysate for pyruvate kinase muscle (PKM), pyruvate kinase liver and red blood cell (PKLR), adenylate kinase 2 (AK2) and glyceraldehyde 3‐phosphatase dehydrogenase (GAPDH). (b) , Immunoblotting of colon tissue lysate for creatine kinase muscle (CKM), adenylate kinase 2 (AK2) and β‐actin. Participant identification numbers are indicated (1001, 1002, 1008, 1012, 1017 and 1020). CGM, cisgender male; TGW, transgender woman.

Techniques Used: Expressing

6) Product Images from "Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation"

Article Title: Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082728

Knockdown of CPSF4 inhibits PI3K/AKT, MAPK signaling and activates caspase-dependent apoptotic pathway in H1299 and A549 cells. ( A ) At 72 hours after siRNA treatment, the expression of CPSF4 protein and the total and phosphorylated Akt, PI3K, ERK1/2, JNK and p38 proteins in H1299 and A549 was detected by Western blot. GAPDH served as the loading control. ( B ) Apoptosis in H1299 and A549 was determined by flow cytometry 72 h after siRNA transfection using an Annexin V-FITC/PI-staining kit. The representative data from three independent experiments are shown. ( C ) Apoptosis was calculated in terms of the FITC-positive in cells. Results are shown as the mean ± SD of three independent experiments (*, P
Figure Legend Snippet: Knockdown of CPSF4 inhibits PI3K/AKT, MAPK signaling and activates caspase-dependent apoptotic pathway in H1299 and A549 cells. ( A ) At 72 hours after siRNA treatment, the expression of CPSF4 protein and the total and phosphorylated Akt, PI3K, ERK1/2, JNK and p38 proteins in H1299 and A549 was detected by Western blot. GAPDH served as the loading control. ( B ) Apoptosis in H1299 and A549 was determined by flow cytometry 72 h after siRNA transfection using an Annexin V-FITC/PI-staining kit. The representative data from three independent experiments are shown. ( C ) Apoptosis was calculated in terms of the FITC-positive in cells. Results are shown as the mean ± SD of three independent experiments (*, P

Techniques Used: Expressing, Western Blot, Flow Cytometry, Cytometry, Transfection, Staining

7) Product Images from "Antiproliferative and apoptotic activity of glycyrrhizinic acid in MCF-7 human breast cancer cells and evaluation of its effect on cell cycle, cell migration and m-TOR/PI3K/Akt signalling pathway"

Article Title: Antiproliferative and apoptotic activity of glycyrrhizinic acid in MCF-7 human breast cancer cells and evaluation of its effect on cell cycle, cell migration and m-TOR/PI3K/Akt signalling pathway

Journal: Archives of Medical Science : AMS

doi: 10.5114/aoms.2018.79429

Glycyrrhizinic acid targets m-TOR, PI3K/Akt pathways in human breast cancer cells. The cells were treated with 0, 10, 50 and 100 µM doses of glycyrrhizinic acid and then the changes in protein expression were evaluated by Western blot assay. GAPDH served as a control
Figure Legend Snippet: Glycyrrhizinic acid targets m-TOR, PI3K/Akt pathways in human breast cancer cells. The cells were treated with 0, 10, 50 and 100 µM doses of glycyrrhizinic acid and then the changes in protein expression were evaluated by Western blot assay. GAPDH served as a control

Techniques Used: Expressing, Western Blot

8) Product Images from "Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma"

Article Title: Histone Deacetylase Inhibitors Antagonize Distinct Pathways to Suppress Tumorigenesis of Embryonal Rhabdomyosarcoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0144320

Trichostatin A (TSA) reduced growth and altered differentiation and self-renewal capacity of ERMS cell lines. (A) Western blots demonstrating hyperacetylation of histones using antibodies against acetyl-histone H3 (Lys9), acetyl-histone H3 (Lys27), acetyl-histone H4 (Lys5), and acetyl-histone H4 (Lys8). D: DMSO; S: vorinostat (SAHA); T: TSA. Each band intensity was normalized to GAPDH loading control. Relative fold increase to DMSO (vehicle) treatment is shown. (B) Analysis of cell viability by cell counts. Cell counts were performed on cells treated with DMSO, 200 nM TSA or 1 μM SAHA at day 0 and day 5. Fold change in cell counts was normalized to day 0. (C-D) Representative images of immunofluorescence (IF) against MF20 performed on RD cells treated with DMSO (C) or 200 nM TSA (D) for 3 days in 2% horse serum in DMEM. Green: MF20-positive cells. Blue: DAPI. Scale bar: 20 μm. (E) Summary of IF against MF20 in ERMS (RD, 381T and SMS-CTR) and ARMS (Rh3, Rh5 and Rh30) cell lines treated with DMSO, 200 nM TSA or 1 μM SAHA. (F) Chromatin immunoprecipitation (ChIP) assays showing differential binding of acetyl-histone H3 (Lys9) at myogenic promoters. Fold enrichment binding of MYOD1 , MYOG , and MYH4 promoter regions were determined by quantitative PCR, normalizing amplification levels to input DNA of each sample. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (G-H) Representative bright-field images from a sphere assay on RD cells treated with DMSO (G) and 200 nM TSA (H). (I) Summary of sphere assays in RD and 381T cells. Each error bar in panels (B), (E), (F) and (I) indicates standard deviation of 3 technical replicates. * indicates p
Figure Legend Snippet: Trichostatin A (TSA) reduced growth and altered differentiation and self-renewal capacity of ERMS cell lines. (A) Western blots demonstrating hyperacetylation of histones using antibodies against acetyl-histone H3 (Lys9), acetyl-histone H3 (Lys27), acetyl-histone H4 (Lys5), and acetyl-histone H4 (Lys8). D: DMSO; S: vorinostat (SAHA); T: TSA. Each band intensity was normalized to GAPDH loading control. Relative fold increase to DMSO (vehicle) treatment is shown. (B) Analysis of cell viability by cell counts. Cell counts were performed on cells treated with DMSO, 200 nM TSA or 1 μM SAHA at day 0 and day 5. Fold change in cell counts was normalized to day 0. (C-D) Representative images of immunofluorescence (IF) against MF20 performed on RD cells treated with DMSO (C) or 200 nM TSA (D) for 3 days in 2% horse serum in DMEM. Green: MF20-positive cells. Blue: DAPI. Scale bar: 20 μm. (E) Summary of IF against MF20 in ERMS (RD, 381T and SMS-CTR) and ARMS (Rh3, Rh5 and Rh30) cell lines treated with DMSO, 200 nM TSA or 1 μM SAHA. (F) Chromatin immunoprecipitation (ChIP) assays showing differential binding of acetyl-histone H3 (Lys9) at myogenic promoters. Fold enrichment binding of MYOD1 , MYOG , and MYH4 promoter regions were determined by quantitative PCR, normalizing amplification levels to input DNA of each sample. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (G-H) Representative bright-field images from a sphere assay on RD cells treated with DMSO (G) and 200 nM TSA (H). (I) Summary of sphere assays in RD and 381T cells. Each error bar in panels (B), (E), (F) and (I) indicates standard deviation of 3 technical replicates. * indicates p

Techniques Used: Western Blot, Immunofluorescence, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Amplification, Negative Control, Standard Deviation

Aberrant NOTCH1-mediated signaling activity is essential for inhibiting myogenic differentiation of ERMS. (A) Summary of MF20 IF of RD cells treated with two different doses of GSI-IX, PD173074, or cyclopamine. (B-C) Representative MF20 IF images of RD cells treated with DMSO (B) and 20 μM GSI-IX (C). Scale bar = 20 μm. (D) Western blot of NOTCH1 expression in RD cells treated with DMSO, 200 nM TSA, or 1 μM SAHA. Each band intensity was normalized to GAPDH loading control. Percent intensity relative to DMSO (vehicle) treatment is shown. (E) Quantitative RT-PCR analysis of NOTCH1 pathway downstream targets, HEY1 , HEY2 and HES1 in RD cells treated with DMSO, 200 nM TSA, or 1 μM SAHA. (F) ChIP assay summary showing differential binding of acetyl-histone H3 (Lys9) at the NOTCH1 promoter in RD cells treated with DMSO or 200 nM TSA. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (G) Summary of MF20 IF of control GFP-overexpressing and NICD-overexpressing RD cells treated with DMSO, 200 nM TSA or 1 μM SAHA. Error bar in each graph indicates standard deviation of technical triplicates. Brackets in each group are used to indicate comparison groups. * indicates p
Figure Legend Snippet: Aberrant NOTCH1-mediated signaling activity is essential for inhibiting myogenic differentiation of ERMS. (A) Summary of MF20 IF of RD cells treated with two different doses of GSI-IX, PD173074, or cyclopamine. (B-C) Representative MF20 IF images of RD cells treated with DMSO (B) and 20 μM GSI-IX (C). Scale bar = 20 μm. (D) Western blot of NOTCH1 expression in RD cells treated with DMSO, 200 nM TSA, or 1 μM SAHA. Each band intensity was normalized to GAPDH loading control. Percent intensity relative to DMSO (vehicle) treatment is shown. (E) Quantitative RT-PCR analysis of NOTCH1 pathway downstream targets, HEY1 , HEY2 and HES1 in RD cells treated with DMSO, 200 nM TSA, or 1 μM SAHA. (F) ChIP assay summary showing differential binding of acetyl-histone H3 (Lys9) at the NOTCH1 promoter in RD cells treated with DMSO or 200 nM TSA. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (G) Summary of MF20 IF of control GFP-overexpressing and NICD-overexpressing RD cells treated with DMSO, 200 nM TSA or 1 μM SAHA. Error bar in each graph indicates standard deviation of technical triplicates. Brackets in each group are used to indicate comparison groups. * indicates p

Techniques Used: Activity Assay, Western Blot, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Standard Deviation

TSA and SAHA reduced tumor growth and induced myogenic differentiation in vivo . (A) Western blots demonstrating hyperacetylation of histone H3 (Lys9) and histone H4 (Lys5) in zebrafish ERMS treated with 1 μM TSA or 50 μM SAHA. GAPDH was used as loading control. The values shown represent fold change in band intensity from TSA or SAHA treatment relative to DMSO after normalizing to loading control. (B) Representative pre- and post-treatment images of zebrafish ERMS treated with DMSO (vehicle) or 1 μM TSA. Dotted line outlines the tumor in each fish. Scale bar = 2 mm. (C) Summary of tumor volume change of zebrafish Tg( myf5 :GFP; mylz2 :mCherry) ERMS treated with DMSO, 50 μM SAHA or 1 μM TSA. Overlaid images of bright field and red fluorescent channel are shown. Error bar indicates standard error of means. n = number of animals treated in each cohort. (D) Summary of quantitative Fluorescence Activated Cell Sorting analysis on ERMS treated with DMSO or 10 μM SAHA. Each pie chart shows relative proportion of each tumor cell subpopulation in an individual treated tumor. Green: myf5 :GFP + / mylz2 :mCherry − cells; yellow: myf5 :GFP + / mylz2 :mCherry + cells; red: mylz2 :mCherry + / myf5 :GFP − cells. (E) Quantitative RT-PCR analysis of myog and myod mRNA expression in ERMS treated with DMSO, 1 μM TSA or 50 μM SAHA. Each bar demonstrates an individual tumor. Each error bar indicates standard deviation of technical triplicates. (F) Annexin V analysis of ERMS tumors treated with DMSO, 50 μM SAHA, or 1μM TSA. 6 animals were analyzed per group. Each error bar indicates standard deviation. * indicates p
Figure Legend Snippet: TSA and SAHA reduced tumor growth and induced myogenic differentiation in vivo . (A) Western blots demonstrating hyperacetylation of histone H3 (Lys9) and histone H4 (Lys5) in zebrafish ERMS treated with 1 μM TSA or 50 μM SAHA. GAPDH was used as loading control. The values shown represent fold change in band intensity from TSA or SAHA treatment relative to DMSO after normalizing to loading control. (B) Representative pre- and post-treatment images of zebrafish ERMS treated with DMSO (vehicle) or 1 μM TSA. Dotted line outlines the tumor in each fish. Scale bar = 2 mm. (C) Summary of tumor volume change of zebrafish Tg( myf5 :GFP; mylz2 :mCherry) ERMS treated with DMSO, 50 μM SAHA or 1 μM TSA. Overlaid images of bright field and red fluorescent channel are shown. Error bar indicates standard error of means. n = number of animals treated in each cohort. (D) Summary of quantitative Fluorescence Activated Cell Sorting analysis on ERMS treated with DMSO or 10 μM SAHA. Each pie chart shows relative proportion of each tumor cell subpopulation in an individual treated tumor. Green: myf5 :GFP + / mylz2 :mCherry − cells; yellow: myf5 :GFP + / mylz2 :mCherry + cells; red: mylz2 :mCherry + / myf5 :GFP − cells. (E) Quantitative RT-PCR analysis of myog and myod mRNA expression in ERMS treated with DMSO, 1 μM TSA or 50 μM SAHA. Each bar demonstrates an individual tumor. Each error bar indicates standard deviation of technical triplicates. (F) Annexin V analysis of ERMS tumors treated with DMSO, 50 μM SAHA, or 1μM TSA. 6 animals were analyzed per group. Each error bar indicates standard deviation. * indicates p

Techniques Used: In Vivo, Western Blot, Fluorescence In Situ Hybridization, Fluorescence, FACS, Quantitative RT-PCR, Expressing, Standard Deviation

EFNB1 is essential for modulating the migratory capacity of ERMS cells. Summary of (A) scratch assays and (B) transwell migration assays on RD and 381T cells with EFNB1 knockdown by two independent siRNAs. Summary of (C) scratch assays and (D) transwell migration assays performed on control GFP-overexpressing and EFNB1-overexpressing RD cell lines. (E) ChIP assays showing differential binding of acetyl-histone H3 (Lys9) on EFNB1 promoter in RD cells treated with DMSO, 200 nM TSA, or 1μM SAHA. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (F) Western blot assessing EFNB1 protein expression level in RD cells treated with DMSO, 200 nM TSA or 1 μM SAHA. Percent intensity relative to DMSO (vehicle) treatment is shown. GAPDH was used as a loading control.
Figure Legend Snippet: EFNB1 is essential for modulating the migratory capacity of ERMS cells. Summary of (A) scratch assays and (B) transwell migration assays on RD and 381T cells with EFNB1 knockdown by two independent siRNAs. Summary of (C) scratch assays and (D) transwell migration assays performed on control GFP-overexpressing and EFNB1-overexpressing RD cell lines. (E) ChIP assays showing differential binding of acetyl-histone H3 (Lys9) on EFNB1 promoter in RD cells treated with DMSO, 200 nM TSA, or 1μM SAHA. Rabbit IgG was used as a negative control for chromatin immunoprecipitation. (F) Western blot assessing EFNB1 protein expression level in RD cells treated with DMSO, 200 nM TSA or 1 μM SAHA. Percent intensity relative to DMSO (vehicle) treatment is shown. GAPDH was used as a loading control.

Techniques Used: Migration, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Western Blot, Expressing

9) Product Images from "The caspase-1 inhibitor AC-YVAD-CMK attenuates acute gastric injury in mice: involvement of silencing NLRP3 inflammasome activities"

Article Title: The caspase-1 inhibitor AC-YVAD-CMK attenuates acute gastric injury in mice: involvement of silencing NLRP3 inflammasome activities

Journal: Scientific Reports

doi: 10.1038/srep24166

Inhibiting caspase-1 reduced cold-restraint stress-induced gastric cell apoptosis. The expression of cleaved caspase-3 and caspase-3 ( A ), and Bax ( B ) proteins was measured by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH. ( C ) Apoptotic cells were observed in the nucleus of the gastric mucosa by TUNEL staining (magnification, 100×). Data are expressed as means ± SD (n = 4). # P
Figure Legend Snippet: Inhibiting caspase-1 reduced cold-restraint stress-induced gastric cell apoptosis. The expression of cleaved caspase-3 and caspase-3 ( A ), and Bax ( B ) proteins was measured by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH. ( C ) Apoptotic cells were observed in the nucleus of the gastric mucosa by TUNEL staining (magnification, 100×). Data are expressed as means ± SD (n = 4). # P

Techniques Used: Expressing, Western Blot, TUNEL Assay, Staining

Inhibiting caspase-1 activity alleviated acute inflammatory responses and improved survival following cold-restraint stress injury. ( A ) The expression of cleaved caspase-1, IL-18, and IL-1β proteins was measured by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH (n = 6). ELISA results showed that mice pretreated with AC-YVAD-CMK had lower levels of TNF-α ( B ) and IL-6 ( C ) compared with those in the injury-alone group (n = 6). ( D ) CD68 mRNA levels in the gastric mucosa were measured by RT-PCR (n = 4). The mean survival times (range) of mice in the injury-alone, injury + omeprazole, and injury + AC-YVAD-CMK groups were 9.08 (8.20–9.95), 11.73 (10.78–12.67), and 10.82 (9.61–12.02), respectively. ( E ) The survival analysis showed that mice pretreated with omeprazole (P
Figure Legend Snippet: Inhibiting caspase-1 activity alleviated acute inflammatory responses and improved survival following cold-restraint stress injury. ( A ) The expression of cleaved caspase-1, IL-18, and IL-1β proteins was measured by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH (n = 6). ELISA results showed that mice pretreated with AC-YVAD-CMK had lower levels of TNF-α ( B ) and IL-6 ( C ) compared with those in the injury-alone group (n = 6). ( D ) CD68 mRNA levels in the gastric mucosa were measured by RT-PCR (n = 4). The mean survival times (range) of mice in the injury-alone, injury + omeprazole, and injury + AC-YVAD-CMK groups were 9.08 (8.20–9.95), 11.73 (10.78–12.67), and 10.82 (9.61–12.02), respectively. ( E ) The survival analysis showed that mice pretreated with omeprazole (P

Techniques Used: Activity Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

Effects of AC-YVAD-CMK on P38/NF-κB signalling pathways. The expression of P-P38 and P38 ( A ), and P-IκB and IκB ( B ) proteins was measured by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH. Data are presented as means ± SD (n = 4). ## P
Figure Legend Snippet: Effects of AC-YVAD-CMK on P38/NF-κB signalling pathways. The expression of P-P38 and P38 ( A ), and P-IκB and IκB ( B ) proteins was measured by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH. Data are presented as means ± SD (n = 4). ## P

Techniques Used: Expressing, Western Blot

The effects of AC-YVAD-CMK pretreatment on the NLRP3 inflammasome in mice. ( A ) The protein levels of NLRP3 and cleaved caspase-1 were determined by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH. ( B ) mRNA levels of ASC were determined by RT-PCR, quantified by densitometry, and expressed as the optical density ratio to GAPDH. Data are expressed as means ± SD (n = 4–6). # P
Figure Legend Snippet: The effects of AC-YVAD-CMK pretreatment on the NLRP3 inflammasome in mice. ( A ) The protein levels of NLRP3 and cleaved caspase-1 were determined by western blotting. Protein levels were quantified by densitometry and are expressed as the optical density ratio to GAPDH. ( B ) mRNA levels of ASC were determined by RT-PCR, quantified by densitometry, and expressed as the optical density ratio to GAPDH. Data are expressed as means ± SD (n = 4–6). # P

Techniques Used: Mouse Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

10) Product Images from "Myeloid-Derived Suppressor Cells Induce Podocyte Injury Through Increasing Reactive Oxygen Species in Lupus Nephritis"

Article Title: Myeloid-Derived Suppressor Cells Induce Podocyte Injury Through Increasing Reactive Oxygen Species in Lupus Nephritis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.01443

Myeloid-derived suppressor cells (MDSCs) induce podocyte injury by reactive oxygen species (ROS). BM-derived MDSCs were pretreated with ROS inhibitors N -acetyl- l -cysteine (5 mM) or inducible nitric oxide synthase inhibitors L-NMMA (0.5 mM) for 1 h, and co-cultured with mouse podocytes at ratio of 1:1 using transwell co-culture systems. (A) Representative light photomicrographs of podocytes after 48 h co-culture with MDSCs. (B) Expression of desmin in podocytes was measured by quantitative real-time RT-PCR after 24 h co-culture with MDSCs. (C,D) Podocyte apoptosis was assessed with annexin V by flow cytometry after 48 h co-culture with MDSCs (C) and the statistic results of podocyte apoptosis (D) . Data represent the mean scores ± SEM. (E) The protein levels of p-p38, p-38, p-p65, and p-65 were assessed by western blot and normalized to GAPDH. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Figure Legend Snippet: Myeloid-derived suppressor cells (MDSCs) induce podocyte injury by reactive oxygen species (ROS). BM-derived MDSCs were pretreated with ROS inhibitors N -acetyl- l -cysteine (5 mM) or inducible nitric oxide synthase inhibitors L-NMMA (0.5 mM) for 1 h, and co-cultured with mouse podocytes at ratio of 1:1 using transwell co-culture systems. (A) Representative light photomicrographs of podocytes after 48 h co-culture with MDSCs. (B) Expression of desmin in podocytes was measured by quantitative real-time RT-PCR after 24 h co-culture with MDSCs. (C,D) Podocyte apoptosis was assessed with annexin V by flow cytometry after 48 h co-culture with MDSCs (C) and the statistic results of podocyte apoptosis (D) . Data represent the mean scores ± SEM. (E) The protein levels of p-p38, p-38, p-p65, and p-65 were assessed by western blot and normalized to GAPDH. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Techniques Used: Derivative Assay, Cell Culture, Co-Culture Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Western Blot

11) Product Images from "Hantaan Virus Nucleocapsid Protein Binds to Importin ? Proteins and Inhibits Tumor Necrosis Factor Alpha-Induced Activation of Nuclear Factor Kappa B ▿"

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

Journal: Journal of Virology

doi: 10.1128/JVI.00986-08

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.
Figure Legend 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.

Techniques Used: 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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

Techniques Used: Expressing, Transfection

12) Product Images from "Non-alcoholic steatohepatitis-related liver tumorigenesis is suppressed in mice lacking hepatic retinoid storage"

Article Title: Non-alcoholic steatohepatitis-related liver tumorigenesis is suppressed in mice lacking hepatic retinoid storage

Journal: Oncotarget

doi: 10.18632/oncotarget.19978

Expression levels of proteins in the livers of the experimental mice Total proteins were extracted from the livers of control and Lrat KO mice, and protein levels of β-catenin and CYP26A1, RARβ, and p21 were examined by western blot analysis, using specific antibodies. GAPDH immunostaining served as the loading control. The bar graph shows the mean intensities of each protein. The values are expressed as the mean ± SD. * P
Figure Legend Snippet: Expression levels of proteins in the livers of the experimental mice Total proteins were extracted from the livers of control and Lrat KO mice, and protein levels of β-catenin and CYP26A1, RARβ, and p21 were examined by western blot analysis, using specific antibodies. GAPDH immunostaining served as the loading control. The bar graph shows the mean intensities of each protein. The values are expressed as the mean ± SD. * P

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

13) Product Images from "HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways"

Article Title: HMGN1 and R848 Synergistically Activate Dendritic Cells Using Multiple Signaling Pathways

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.02982

N1 and R848 synergistically activate IRF signaling as well as type 1 IFN responses in human MoDCs. (A,B) Serum-starved human MoDCs (2 × 10 6 cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in buffer (2 × 10 6 MoDCs/0.1 ml) for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of (A) cytoplasmic fraction of IRF3, GAPDH, and nuclear fraction of IRF3, lamin B1 or (B) cytoplasmic fraction of IRF7, GAPDH, and nuclear fraction of IRF7, lamin B1. Band intensities from three donors was quantified and normalized with GAPDH or lamin B1 using ImageJ software. (C–E) Human MoDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before extraction of total RNA. The levels of IFN-α2, IFN-α4, and IFN-β1 mRNA were quantitated by qPCR and shown as a fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. * p
Figure Legend Snippet: N1 and R848 synergistically activate IRF signaling as well as type 1 IFN responses in human MoDCs. (A,B) Serum-starved human MoDCs (2 × 10 6 cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in buffer (2 × 10 6 MoDCs/0.1 ml) for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of (A) cytoplasmic fraction of IRF3, GAPDH, and nuclear fraction of IRF3, lamin B1 or (B) cytoplasmic fraction of IRF7, GAPDH, and nuclear fraction of IRF7, lamin B1. Band intensities from three donors was quantified and normalized with GAPDH or lamin B1 using ImageJ software. (C–E) Human MoDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before extraction of total RNA. The levels of IFN-α2, IFN-α4, and IFN-β1 mRNA were quantitated by qPCR and shown as a fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. * p

Techniques Used: Translocation Assay, Electrophoresis, Software, Real-time Polymerase Chain Reaction

N1 and R848 synergistically activate NF-κB, IRF3, and IRF7 signaling as well as type 1 IFN responses in mouse BMDCs. (A–C) Mouse BMDCs (2 × 10 6 cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in the buffer for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of proteins was loaded and separated by electrophoresis to detect the levels of cytoplasmic fraction of IRF3, IRF7, phospho-p65, and GAPDH, as well as nuclear fraction of IRF3, IRF7, phospho-p65, and lamin B1. Quantitation of band intensities from three different batches of male mice ( n = 6) were normalized against GAPDH or lamin B1 using ImageJ software. (D) Mouse BMDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before being used for the extraction of total RNA. The levels of IFN-β1 mRNA was quantitated by qPCR and shown as fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. * p
Figure Legend Snippet: N1 and R848 synergistically activate NF-κB, IRF3, and IRF7 signaling as well as type 1 IFN responses in mouse BMDCs. (A–C) Mouse BMDCs (2 × 10 6 cells/ml) were treated for 0–90 min with N1 (500 ng/ml) and R848 (500 ng/ml) before they were solubilized in the buffer for analysis of the translocation of proteins from cytoplasmic to nuclear cell fractions. An identical amount of proteins was loaded and separated by electrophoresis to detect the levels of cytoplasmic fraction of IRF3, IRF7, phospho-p65, and GAPDH, as well as nuclear fraction of IRF3, IRF7, phospho-p65, and lamin B1. Quantitation of band intensities from three different batches of male mice ( n = 6) were normalized against GAPDH or lamin B1 using ImageJ software. (D) Mouse BMDCs were treated with N1 (500 ng/ml) and/or R848 (500 ng/ml) for 6 h before being used for the extraction of total RNA. The levels of IFN-β1 mRNA was quantitated by qPCR and shown as fold increase over the sham treated DCs. Data are shown as the average (mean ± SD) of triplicates of one experiment representative of three. * p

Techniques Used: Translocation Assay, Electrophoresis, Quantitation Assay, Mouse Assay, Software, Real-time Polymerase Chain Reaction

N1 and R848 synergistically activate NF-κB and MAPK signaling in human MoDCs. Serum-starved human MoDCs (2 × 10 6 cells/ml) were treated for 0–90 min with N1 (250 ng/ml) and R848 (250 ng/ml) before they were solubilized in lysis buffer (10 6 MoDCs/0.1 ml) to obtain whole cell lysates or in buffer (2 × 10 6 MoDCs/0.1 ml) for analysis of the translocation of proteins from the cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of phospho-I-κBα, I-κBα, phospho-p65, phospho-p38, p38, phospho-CREB, CREB, phospho-JNK, JNK, phospho-c-Jun, c-Jun, and GAPDH in whole cell lysates (A,C) or (B) cytoplasmic fraction of phospho-p65, GAPDH and nuclear fraction of phospho-p65, lamin B1. Quantitation of band intensities from three donors using ImageJ software were normalized against GAPDH or lamin B1. ** p
Figure Legend Snippet: N1 and R848 synergistically activate NF-κB and MAPK signaling in human MoDCs. Serum-starved human MoDCs (2 × 10 6 cells/ml) were treated for 0–90 min with N1 (250 ng/ml) and R848 (250 ng/ml) before they were solubilized in lysis buffer (10 6 MoDCs/0.1 ml) to obtain whole cell lysates or in buffer (2 × 10 6 MoDCs/0.1 ml) for analysis of the translocation of proteins from the cytoplasmic to nuclear cell fractions. An identical amount of protein was loaded and separated by electrophoresis to detect the levels of phospho-I-κBα, I-κBα, phospho-p65, phospho-p38, p38, phospho-CREB, CREB, phospho-JNK, JNK, phospho-c-Jun, c-Jun, and GAPDH in whole cell lysates (A,C) or (B) cytoplasmic fraction of phospho-p65, GAPDH and nuclear fraction of phospho-p65, lamin B1. Quantitation of band intensities from three donors using ImageJ software were normalized against GAPDH or lamin B1. ** p

Techniques Used: Lysis, Translocation Assay, Electrophoresis, Quantitation Assay, Software

14) Product Images from "Design, Synthesis and Structure-Activity Relationship Studies of Novel Survivin Inhibitors with Potent Anti-Proliferative Properties"

Article Title: Design, Synthesis and Structure-Activity Relationship Studies of Novel Survivin Inhibitors with Potent Anti-Proliferative Properties

Journal: PLoS ONE

doi: 10.1371/journal.pone.0129807

Representative drug affinity responsive target stability (DARTS) results for pronase-digested A375 or M14 cell lysates. Immunoblotting showed protection of the target protein, survivin, by incubation with compound 4g at the concentration of 20 μM, whereas digestion of the non-target proteins like GAPDH was unchanged.
Figure Legend Snippet: Representative drug affinity responsive target stability (DARTS) results for pronase-digested A375 or M14 cell lysates. Immunoblotting showed protection of the target protein, survivin, by incubation with compound 4g at the concentration of 20 μM, whereas digestion of the non-target proteins like GAPDH was unchanged.

Techniques Used: Incubation, Concentration Assay

15) Product Images from "Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate and breast carcinoma cells"

Article Title: Inhibitors of histone deacetylase 1 reverse the immune evasion phenotype to enhance T-cell mediated lysis of prostate and breast carcinoma cells

Journal: Oncotarget

doi: 10.18632/oncotarget.7180

Vorinostat-induced immunogenic modulation is mediated by the unfolded protein response MDA-MB-231 cells were exposed to siRNA control or targeting ERN1 or PERK for 24 h prior to being exposed to vehicle (DMSO) or vorinostat (3 μM) as described in Materials and Methods. A. and B. At the end of treatment, total cell lysates were examined by Western blotting to determine expression of ERN1 (A) or PERK (B). GAPDH was used as internal control for total protein levels. Silencing ratio denotes target protein expression relative to GAPDH, further normalized to protein levels after treatment in the presence of silencing RNA control. C. and D. At the end of treatment, MDA-MB-231 cells were used as targets in a CTL lysis assay using CEA-specific CD8 + T cells as effectors (E:T = 30:1). Results are presented as mean ± S.E.M. from 6 replicate wells, and are representative of 2-3 independent experiments. Asterisks denote statistical significance relative to controls ( P
Figure Legend Snippet: Vorinostat-induced immunogenic modulation is mediated by the unfolded protein response MDA-MB-231 cells were exposed to siRNA control or targeting ERN1 or PERK for 24 h prior to being exposed to vehicle (DMSO) or vorinostat (3 μM) as described in Materials and Methods. A. and B. At the end of treatment, total cell lysates were examined by Western blotting to determine expression of ERN1 (A) or PERK (B). GAPDH was used as internal control for total protein levels. Silencing ratio denotes target protein expression relative to GAPDH, further normalized to protein levels after treatment in the presence of silencing RNA control. C. and D. At the end of treatment, MDA-MB-231 cells were used as targets in a CTL lysis assay using CEA-specific CD8 + T cells as effectors (E:T = 30:1). Results are presented as mean ± S.E.M. from 6 replicate wells, and are representative of 2-3 independent experiments. Asterisks denote statistical significance relative to controls ( P

Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, CTL Assay, Lysis

Vorinostat-induced immunogenic modulation of MDA-MB-231 carcinoma cells is mediated by HDAC1 MDA-MB-231 cells were exposed to siRNA control or targeting HDAC1 for 24 h prior to being exposed to vehicle (DMSO) or vorinostat (3 μM) as described in Materials and Methods. A. At the end of treatment, total cell lysates were examined by Western blotting to determine expression of HDAC1. GAPDH was used as internal control for total protein levels. Silencing ratio denotes HDAC1 protein expression relative to GAPDH, further normalized to protein levels after treatment in the presence of silencing RNA control. B. At the end of treatment, MDA-MB-231 cells were used as targets in a CTL-lysis assay where effector brachyury-specific CD8 + T cells were used at an E:T ratio of 30:1. Results are presented as mean ± S.E.M. from 4-6 replicate wells. Asterisks denote statistical significance relative to controls ( *P = 0.002). Data is representative of two independent experiments.
Figure Legend Snippet: Vorinostat-induced immunogenic modulation of MDA-MB-231 carcinoma cells is mediated by HDAC1 MDA-MB-231 cells were exposed to siRNA control or targeting HDAC1 for 24 h prior to being exposed to vehicle (DMSO) or vorinostat (3 μM) as described in Materials and Methods. A. At the end of treatment, total cell lysates were examined by Western blotting to determine expression of HDAC1. GAPDH was used as internal control for total protein levels. Silencing ratio denotes HDAC1 protein expression relative to GAPDH, further normalized to protein levels after treatment in the presence of silencing RNA control. B. At the end of treatment, MDA-MB-231 cells were used as targets in a CTL-lysis assay where effector brachyury-specific CD8 + T cells were used at an E:T ratio of 30:1. Results are presented as mean ± S.E.M. from 4-6 replicate wells. Asterisks denote statistical significance relative to controls ( *P = 0.002). Data is representative of two independent experiments.

Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing, CTL Assay, Lysis

16) Product Images from "Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway"

Article Title: Zuo Jin Wan reverses P-gp-mediated drug-resistance by inhibiting activation of the PI3K/Akt/NF-κB pathway

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/1472-6882-14-279

ZJW suppresses P-gp mediated MDR by inhibiting activation of Akt (Ser473)/IκB phosphorylation (Ser473) in vitro . (A B) Western blotting assay was carried out to detect the level of Akt, phosphorylation of Akt (Thr307/Ser473), IκB, p65, and phosphorylation of IκB in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. Data are means ± SD of values from triplicate experiments. ** P
Figure Legend Snippet: ZJW suppresses P-gp mediated MDR by inhibiting activation of Akt (Ser473)/IκB phosphorylation (Ser473) in vitro . (A B) Western blotting assay was carried out to detect the level of Akt, phosphorylation of Akt (Thr307/Ser473), IκB, p65, and phosphorylation of IκB in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. Data are means ± SD of values from triplicate experiments. ** P

Techniques Used: Activation Assay, In Vitro, Western Blot

ZJW inhibits P-gp expression and the effect of the PI3K/Akt pathway. (A) Western blotting assay was carried out to detect the level of P-gp, LRP, and MRP-2 in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. (B) Blots were photographed and quantitated; the data are from three independent experiments. (C) Real-time PCR was performed to detect ABCB1 mRNA in vivo . Quantification of ABCB1 mRNA was performed by assigning a value of 1 to the control group treatment with ZJW (50 μg/mL). Statistical difference was analyzed by student’s t -test, ** P
Figure Legend Snippet: ZJW inhibits P-gp expression and the effect of the PI3K/Akt pathway. (A) Western blotting assay was carried out to detect the level of P-gp, LRP, and MRP-2 in HCT116/L-OHP cells treated with LY294002 (20 μM, 2 h), ZJW (50 μg/mL, 48 h), and a combination of ZJW (50 μg/mL, 48 h) and IGF-1 (100 ng/mL, 48 h). GAPDH was used to ensure equal loading of proteins in each lane. (B) Blots were photographed and quantitated; the data are from three independent experiments. (C) Real-time PCR was performed to detect ABCB1 mRNA in vivo . Quantification of ABCB1 mRNA was performed by assigning a value of 1 to the control group treatment with ZJW (50 μg/mL). Statistical difference was analyzed by student’s t -test, ** P

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

17) Product Images from "LINC01133 as ceRNA inhibits gastric cancer progression by sponging miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway"

Article Title: LINC01133 as ceRNA inhibits gastric cancer progression by sponging miR-106a-3p to regulate APC expression and the Wnt/β-catenin pathway

Journal: Molecular Cancer

doi: 10.1186/s12943-018-0874-1

LINC01133 attenuates the EMT and metastatic abilities of GC cells through APC/Wnt signaling pathway. a Identification of 93 commonly changed targeted mRNAs of miR-106a-3p from the four publicly profile datasets (TargetScan, microT-CDS, TargetMiner, and mirDIP). Different color areas represented different datasets. The cross areas meant the commonly changed mRNAs of miR-106a-3p. b GO analysis and significant enriched GO terms of 93 commonly changed targeted mRNAs in gastric cancer on their biological process (BP). The statistically significant results were defined with -log10 ( P value) > 1.30 as the cut-off criterion. c , d Dual luciferase assay demonstrating the effect on TOP/FOP reporter activity in HEK-293FT cells, AGS cells transfected with shLINC01133 vector or SGC-7901 cells with LINC01133 overexpression. Results were normalized to a Renilla transfection control. e Dual luciferase assay showing the effect on TOP/FOP reporter activity in AGS cells following reduced expression of LINC01133 and/or inhibition of miR-106a-3p. f Immunoblot assay of E-cadherin, vimentin, N-cadherin, APC, and total and nuclear β-catenin proteins in AGS cells transfected with shLINC01133–2 and/or miR-106a-3p inhibitor. Numbers showed quantification of relative protein amount. GAPDH was used as an internal control. Lamin B1 was used as an endogenous control for the cell nuclear fraction. g Schematic diagram of the regulatory mechanism of LINC01133/miR-106a-3p/APC axis in the inhibition of GC proliferation and metastasis. Error bars: mean ± SD, n = 3. * P
Figure Legend Snippet: LINC01133 attenuates the EMT and metastatic abilities of GC cells through APC/Wnt signaling pathway. a Identification of 93 commonly changed targeted mRNAs of miR-106a-3p from the four publicly profile datasets (TargetScan, microT-CDS, TargetMiner, and mirDIP). Different color areas represented different datasets. The cross areas meant the commonly changed mRNAs of miR-106a-3p. b GO analysis and significant enriched GO terms of 93 commonly changed targeted mRNAs in gastric cancer on their biological process (BP). The statistically significant results were defined with -log10 ( P value) > 1.30 as the cut-off criterion. c , d Dual luciferase assay demonstrating the effect on TOP/FOP reporter activity in HEK-293FT cells, AGS cells transfected with shLINC01133 vector or SGC-7901 cells with LINC01133 overexpression. Results were normalized to a Renilla transfection control. e Dual luciferase assay showing the effect on TOP/FOP reporter activity in AGS cells following reduced expression of LINC01133 and/or inhibition of miR-106a-3p. f Immunoblot assay of E-cadherin, vimentin, N-cadherin, APC, and total and nuclear β-catenin proteins in AGS cells transfected with shLINC01133–2 and/or miR-106a-3p inhibitor. Numbers showed quantification of relative protein amount. GAPDH was used as an internal control. Lamin B1 was used as an endogenous control for the cell nuclear fraction. g Schematic diagram of the regulatory mechanism of LINC01133/miR-106a-3p/APC axis in the inhibition of GC proliferation and metastasis. Error bars: mean ± SD, n = 3. * P

Techniques Used: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Over Expression, Expressing, Inhibition

LINC01133 inhibits proliferation, migration and EMT in vitro and in vivo. a Relative expression of LINC01133 confirmed by qRT-PCR in SGC-7901 and HGC-27 cells with LINC01133 overexpression. b , c Proliferation and migration assays of SGC-7901 and HGC-27 cells with LINC01133 overexpression by the CCK-8 assay ( b) , and transwell assay ( c) . d Immunoblot assay to evaluate the expression of vimentin, E-cadherin, and N-cadherin proteins in AGS and BGC-823 cells after LINC01133 knockdown or in SGC-7901 cells after LINC01133 overexpression. The reference protein GAPDH was used as an internal control. Numbers shows the ratio of target protein/GAPDH (arbitrary unit). e The right armpit was injected with SGC-7901 cells transfected with LINC01133 expression vector or empty vector in upper panel. Representative images of xenograft tumors are indicated in the bottom panel. f , g Tumor volume and weight of the xenograft in LINC01133 overexpression groups and control group. h Left panel: representative images of lung metastatic nodules of LINC01133 overexpression groups and control group are indicated by blue arrows. Representative hematoxylin and eosin (H E) staining results of corresponding lung metastatic nodules are shown in right panel. i Statistical analysis of numbers of metastatic nodules in the lung. Error bars: mean ± SD from three independent experiments. * P
Figure Legend Snippet: LINC01133 inhibits proliferation, migration and EMT in vitro and in vivo. a Relative expression of LINC01133 confirmed by qRT-PCR in SGC-7901 and HGC-27 cells with LINC01133 overexpression. b , c Proliferation and migration assays of SGC-7901 and HGC-27 cells with LINC01133 overexpression by the CCK-8 assay ( b) , and transwell assay ( c) . d Immunoblot assay to evaluate the expression of vimentin, E-cadherin, and N-cadherin proteins in AGS and BGC-823 cells after LINC01133 knockdown or in SGC-7901 cells after LINC01133 overexpression. The reference protein GAPDH was used as an internal control. Numbers shows the ratio of target protein/GAPDH (arbitrary unit). e The right armpit was injected with SGC-7901 cells transfected with LINC01133 expression vector or empty vector in upper panel. Representative images of xenograft tumors are indicated in the bottom panel. f , g Tumor volume and weight of the xenograft in LINC01133 overexpression groups and control group. h Left panel: representative images of lung metastatic nodules of LINC01133 overexpression groups and control group are indicated by blue arrows. Representative hematoxylin and eosin (H E) staining results of corresponding lung metastatic nodules are shown in right panel. i Statistical analysis of numbers of metastatic nodules in the lung. Error bars: mean ± SD from three independent experiments. * P

Techniques Used: Migration, In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Over Expression, CCK-8 Assay, Transwell Assay, Injection, Transfection, Plasmid Preparation, Staining

18) Product Images from "MiR-23a transcriptional activated by Runx2 increases metastatic potential of mouse hepatoma cell via directly targeting Mgat3"

Article Title: MiR-23a transcriptional activated by Runx2 increases metastatic potential of mouse hepatoma cell via directly targeting Mgat3

Journal: Scientific Reports

doi: 10.1038/s41598-018-25768-z

miR-23a is positively and direct regulated by Runx2. ( a ) Runx2 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (lower). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( b ) The putative Runx2-binding sequence (E-BOX) in the miR-23a gene promoter is shown. A mutation was generated in the miR-23a promoter at the complementary site for the E-BOX of Runx2 (red). ( c ) Luciferase assays of reporter activity after co-transfection of either the partial miR-23a promoter or the mutant promoter with the pCMV-Runx2 plasmid into Hepa1–6 cells. ( d ) ChIP assays were performed according to the EZ-Magna ChIP TM .
Figure Legend Snippet: miR-23a is positively and direct regulated by Runx2. ( a ) Runx2 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (lower). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( b ) The putative Runx2-binding sequence (E-BOX) in the miR-23a gene promoter is shown. A mutation was generated in the miR-23a promoter at the complementary site for the E-BOX of Runx2 (red). ( c ) Luciferase assays of reporter activity after co-transfection of either the partial miR-23a promoter or the mutant promoter with the pCMV-Runx2 plasmid into Hepa1–6 cells. ( d ) ChIP assays were performed according to the EZ-Magna ChIP TM .

Techniques Used: Expressing, High Content Screening, Western Blot, Quantitative RT-PCR, Binding Assay, Sequencing, Mutagenesis, Generated, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Chromatin Immunoprecipitation

Mgat3 is a direct target of miR-23a. ( a . A mutation was generated in the Mgat3 3′UTR sequence at the complementary site for the seed region of miR-23a (red). ( b ) The luciferase reporter assays show reporter activity after co-transfection of either psiCHECK-2-Mgat3-3′UTR (2180–2751 bp) or psiCHECK-2-mutant Mgat3 3′UTR with miR-23a mimic in Hepa1–6 cells. ( c , d ) miR-23a and Mgat3 expression levels in mouse HCC cells transfected with miR-23a mimic or miR-23a inhibitor relative to those transfected with CP transfection reagent only (mock) or scrambled miR-23a (nc) as measured by qRT-PCR and Western blotting. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p
Figure Legend Snippet: Mgat3 is a direct target of miR-23a. ( a . A mutation was generated in the Mgat3 3′UTR sequence at the complementary site for the seed region of miR-23a (red). ( b ) The luciferase reporter assays show reporter activity after co-transfection of either psiCHECK-2-Mgat3-3′UTR (2180–2751 bp) or psiCHECK-2-mutant Mgat3 3′UTR with miR-23a mimic in Hepa1–6 cells. ( c , d ) miR-23a and Mgat3 expression levels in mouse HCC cells transfected with miR-23a mimic or miR-23a inhibitor relative to those transfected with CP transfection reagent only (mock) or scrambled miR-23a (nc) as measured by qRT-PCR and Western blotting. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p

Techniques Used: Mutagenesis, Generated, Sequencing, Luciferase, Activity Assay, Cotransfection, Expressing, Transfection, Quantitative RT-PCR, Western Blot

Runx2 regulates Mgat3 expression via miR-23a. ( a ) Runx2, miR-23a and Mgat3 expression levels were measured by qRT-PCR (upper) and Western blot (lower) after transfection of 3.6 µg of pCMV-Runx2 plasmid or 150 nM miR-23a inhibitor and 3 µg of pCMV-Runx2 plasmid into Hepa1–6 cells relative to transfection of CP transfection reagent only (mock). ( b ) Runx2, miR-23a and Mgat3 mRNA levels were determined in Hca-P cells transfected with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( c ) FCM analysis of the levels of bisecting structures on the cell surface recognized by FITC-PHA-E after transfection of Hca-P cells with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. ( d for additional FCM analysis of the levels of β-1,6 branching of N-glycans. Data are presented as median with error bars (*p
Figure Legend Snippet: Runx2 regulates Mgat3 expression via miR-23a. ( a ) Runx2, miR-23a and Mgat3 expression levels were measured by qRT-PCR (upper) and Western blot (lower) after transfection of 3.6 µg of pCMV-Runx2 plasmid or 150 nM miR-23a inhibitor and 3 µg of pCMV-Runx2 plasmid into Hepa1–6 cells relative to transfection of CP transfection reagent only (mock). ( b ) Runx2, miR-23a and Mgat3 mRNA levels were determined in Hca-P cells transfected with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. ( c ) FCM analysis of the levels of bisecting structures on the cell surface recognized by FITC-PHA-E after transfection of Hca-P cells with 100 nM Runx2 siRNA, 100 nM Runx2 siRNA and 3 µg of pCMV-Runx2 plasmid (Runx2 rescued after 24 h), or 150 nM miR-23a mimic and 100 nM Runx2 siRNA relative to the mock control. ( d for additional FCM analysis of the levels of β-1,6 branching of N-glycans. Data are presented as median with error bars (*p

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, High Content Screening

Constitutive expression of miR-23a and Mgat3 in mouse HCC cell lines. ( a , measured by microarray is displayed as a histogram (right). ( b ) The relative expression of miR-23a in Hca-P and Hepa1–6 cells as measured by qRT-PCR. ( c ) Mgat3 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (below). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p
Figure Legend Snippet: Constitutive expression of miR-23a and Mgat3 in mouse HCC cell lines. ( a , measured by microarray is displayed as a histogram (right). ( b ) The relative expression of miR-23a in Hca-P and Hepa1–6 cells as measured by qRT-PCR. ( c ) Mgat3 expression level in Hca-P and Hepa1–6 cells, as measured by Western blotting (upper) and qRT-PCR (below). The numbers upside WB figure present relative intensity of the bands normalized by corresponding GAPDH bands. Data are presented as the median with error bars (*p

Techniques Used: Expressing, Microarray, High Content Screening, Quantitative RT-PCR, Western Blot

19) Product Images from "LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis"

Article Title: LRP-1 Promotes Colon Cancer Cell Proliferation in 3D Collagen Matrices by Mediating DDR1 Endocytosis

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00412

RAP treatment inhibits DDR1 endocytosis and led to its accumulation at the plasma membrane. (A) Plasma membrane extracts from cell surface biotinylated proteins were obtained from HT-29 cells treated or not with RAP (1 μM, 1 h). Immunoblot analysis was performed using anti-DDR1 antibodies. Expression level of GAPDH in the intracellular fraction served as a loading control and for normalization. Three independent experiments were conducted, the data is represented as the mean SD. ** p
Figure Legend Snippet: RAP treatment inhibits DDR1 endocytosis and led to its accumulation at the plasma membrane. (A) Plasma membrane extracts from cell surface biotinylated proteins were obtained from HT-29 cells treated or not with RAP (1 μM, 1 h). Immunoblot analysis was performed using anti-DDR1 antibodies. Expression level of GAPDH in the intracellular fraction served as a loading control and for normalization. Three independent experiments were conducted, the data is represented as the mean SD. ** p

Techniques Used: Expressing

Molecular characterization of colorectal carcinoma cell lines. (A) Transcriptional level of LRP-1 and DDR1 were assessed using RTqPCR. LRP-1 and DDR1 mRNA expression levels in HT-29 (black boxes) and LS174T (gray boxes) were normalized with both RPL32 and RS18 mRNA expression. (B) Whole cell extracts from HT-29 and LS174T cells were analyzed by SDS PAGE followed by western blotting using anti-DDR1, anti-LRP-1 and anti-GAPDH antibodies. Graphical representations of LRP-1 (C) and DDR1 (D) expression at protein level as normalized with GAPDH. All experiments were performed in three biological replicates. Plots are presented as the mean SD, ** p
Figure Legend Snippet: Molecular characterization of colorectal carcinoma cell lines. (A) Transcriptional level of LRP-1 and DDR1 were assessed using RTqPCR. LRP-1 and DDR1 mRNA expression levels in HT-29 (black boxes) and LS174T (gray boxes) were normalized with both RPL32 and RS18 mRNA expression. (B) Whole cell extracts from HT-29 and LS174T cells were analyzed by SDS PAGE followed by western blotting using anti-DDR1, anti-LRP-1 and anti-GAPDH antibodies. Graphical representations of LRP-1 (C) and DDR1 (D) expression at protein level as normalized with GAPDH. All experiments were performed in three biological replicates. Plots are presented as the mean SD, ** p

Techniques Used: Expressing, SDS Page, Western Blot

20) Product Images from "HDAC6 promotes self-renewal and migration/invasion of rhabdomyosarcoma"

Article Title: HDAC6 promotes self-renewal and migration/invasion of rhabdomyosarcoma

Journal: bioRxiv

doi: 10.1101/823864

HDAC6 expression in RMS and conserved function of HDAC6 in regulating RMS tumor growth. (A-E) Immunohistochemistry for HDAC6 in representative primary ERMS and ARMS tumors and skeletal muscle tissue. Weaker staining in (A-B) and stronger staining in (C-D). No staining is seen in skeletal muscle (E). (F) Summary of IHC results in ERMS (n = 7), ARMS (n = 11) and skeletal muscle (n = 5). Immuofluorescence for HDAC6 in (G-H) ERMS cell lines (RD and SMS-CTR) and (I and J) ARMS cell lines (Rh5 and Rh30). (K) Summary of change in cell growth by cell counts following HDAC6 knockout (KO) by CRISPR/Cas9 in a panel of RMS cell lines. The results shown are the average of 3 replicates for each cell line from one of 3 independent experiments. (L) Overexpression of GFP as control, Cas9-resistant wild-type (wt) HDAC6 and Cas9-resistant catalytically-dead (cd) HDAC6 in tamoxifen (TAM)-inducible Cas9/ HDAC6 gRNA RD line to assess change in cell growth following TAM-induction. Results shown are the average of 4 replicates from one of 3 independent experiments. (M) Western blots against HDAC6 in RD and Rh5 cells with targeted disruption of HDAC6 by CRISPR (left panel) and in RD TAM-inducible Cas9/ HDAC6 gRNA line overexpressing GFP, wt HDAC6 and cd HDAC6 (right panel). HDAC6 g = HDAC6 gRNA. GAPDH was used as a loading control. (N) A snapshot of genomic DNA sequencing results demonstrating deletion in the hdac6 locus in a CRISPR-targeted zebrafish tumor. (O-R) Representative zebrafish ERMS tumors expressing GFP control vector (O, Q) and Cas9/ hdac6 gRNA (P, R) over 7 days of growth. (S) Summary of tumor growth. n = 8 for control and n = 6 for hdac6 gRNA-targeted ERMS tumors. Each error bar in graphs of K, L and S represents standard deviation. ** = p
Figure Legend Snippet: HDAC6 expression in RMS and conserved function of HDAC6 in regulating RMS tumor growth. (A-E) Immunohistochemistry for HDAC6 in representative primary ERMS and ARMS tumors and skeletal muscle tissue. Weaker staining in (A-B) and stronger staining in (C-D). No staining is seen in skeletal muscle (E). (F) Summary of IHC results in ERMS (n = 7), ARMS (n = 11) and skeletal muscle (n = 5). Immuofluorescence for HDAC6 in (G-H) ERMS cell lines (RD and SMS-CTR) and (I and J) ARMS cell lines (Rh5 and Rh30). (K) Summary of change in cell growth by cell counts following HDAC6 knockout (KO) by CRISPR/Cas9 in a panel of RMS cell lines. The results shown are the average of 3 replicates for each cell line from one of 3 independent experiments. (L) Overexpression of GFP as control, Cas9-resistant wild-type (wt) HDAC6 and Cas9-resistant catalytically-dead (cd) HDAC6 in tamoxifen (TAM)-inducible Cas9/ HDAC6 gRNA RD line to assess change in cell growth following TAM-induction. Results shown are the average of 4 replicates from one of 3 independent experiments. (M) Western blots against HDAC6 in RD and Rh5 cells with targeted disruption of HDAC6 by CRISPR (left panel) and in RD TAM-inducible Cas9/ HDAC6 gRNA line overexpressing GFP, wt HDAC6 and cd HDAC6 (right panel). HDAC6 g = HDAC6 gRNA. GAPDH was used as a loading control. (N) A snapshot of genomic DNA sequencing results demonstrating deletion in the hdac6 locus in a CRISPR-targeted zebrafish tumor. (O-R) Representative zebrafish ERMS tumors expressing GFP control vector (O, Q) and Cas9/ hdac6 gRNA (P, R) over 7 days of growth. (S) Summary of tumor growth. n = 8 for control and n = 6 for hdac6 gRNA-targeted ERMS tumors. Each error bar in graphs of K, L and S represents standard deviation. ** = p

Techniques Used: Expressing, Immunohistochemistry, Staining, Knock-Out, CRISPR, Over Expression, Western Blot, DNA Sequencing, Plasmid Preparation, Standard Deviation

21) Product Images from "Functional Differences Between IgM and IgD Signaling in Chronic Lymphocytic Leukemia"

Article Title: Functional Differences Between IgM and IgD Signaling in Chronic Lymphocytic Leukemia

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1600915

CCL3 and CCL4 chemokine secretion and BCL6 protein expression analyses (A) CCL3 and (B) CCL4 measurement by ELISA in the supernatants of 7 U-CLL samples after a 24-hour stimulation with increasing concentrations of anti-IgM and anti-IgD. Displayed are the means ± SEM of chemokine concentrations in pg/mL. (C) Western Blot analysis of BCL6 protein expression at serial time points following anti-IgM or anti-IgD of a U-CLL case. GAPDH protein levels were analyzed, as loading control. A representative U-CLL case out of 3 is shown. (D) Quantification of BCL6 protein expression after anti-IgM or anti-IgD in 3 U-CLL cases, expressed as normalized intensity between BCL6 and GAPDH levels. ** P
Figure Legend Snippet: CCL3 and CCL4 chemokine secretion and BCL6 protein expression analyses (A) CCL3 and (B) CCL4 measurement by ELISA in the supernatants of 7 U-CLL samples after a 24-hour stimulation with increasing concentrations of anti-IgM and anti-IgD. Displayed are the means ± SEM of chemokine concentrations in pg/mL. (C) Western Blot analysis of BCL6 protein expression at serial time points following anti-IgM or anti-IgD of a U-CLL case. GAPDH protein levels were analyzed, as loading control. A representative U-CLL case out of 3 is shown. (D) Quantification of BCL6 protein expression after anti-IgM or anti-IgD in 3 U-CLL cases, expressed as normalized intensity between BCL6 and GAPDH levels. ** P

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

22) Product Images from "S-Propargyl-Cysteine, a Novel Water-Soluble Modulator of Endogenous Hydrogen Sulfide, Promotes Angiogenesis Through Activation of Signal Transducer and Activator of Transcription 3"

Article Title: S-Propargyl-Cysteine, a Novel Water-Soluble Modulator of Endogenous Hydrogen Sulfide, Promotes Angiogenesis Through Activation of Signal Transducer and Activator of Transcription 3

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2013.5449

SPRC induced the nuclear translocation of STAT3, followed by transcriptional activation. (A) HUV-EC-C were treated with SPRC (100 μ M ) or interferon (IFN)-γ (100 ng/ml) for 45 min, and then, cytoplasmic and nuclear proteins were isolated for Western blotting. GAPDH and histone H3 were used as a loading control for cytoplasmic and nuclear proteins, respectively. (B) Primary HUVEC were treated with SPRC (100 μ M ) for 45 min and then stained with specific antibody (red) or DAPI (blue). Scale bar, 20 μm. (C) HUV-EC-C were treated with SPRC (100 μ M ), IFN-γ (100 ng/ml), or WP1066 (10 μ M ) for 45 min. The nuclear extract was isolated and used for electrophoretic mobility shift assay. The shift band of STAT3 is indicated with a solid arrowhead . IFN-γ was used as a positive control, and WP1066 was used to inhibit STAT3. (D) HUV-EC-C were treated with SPRC (100 μ M ) for 45 min. The nuclear extract was isolated and used to perform chromatin immunoprecipitation with an STAT3 antibody. The change of downstream promoters was analyzed by real-time polymerase chain reaction. # p
Figure Legend Snippet: SPRC induced the nuclear translocation of STAT3, followed by transcriptional activation. (A) HUV-EC-C were treated with SPRC (100 μ M ) or interferon (IFN)-γ (100 ng/ml) for 45 min, and then, cytoplasmic and nuclear proteins were isolated for Western blotting. GAPDH and histone H3 were used as a loading control for cytoplasmic and nuclear proteins, respectively. (B) Primary HUVEC were treated with SPRC (100 μ M ) for 45 min and then stained with specific antibody (red) or DAPI (blue). Scale bar, 20 μm. (C) HUV-EC-C were treated with SPRC (100 μ M ), IFN-γ (100 ng/ml), or WP1066 (10 μ M ) for 45 min. The nuclear extract was isolated and used for electrophoretic mobility shift assay. The shift band of STAT3 is indicated with a solid arrowhead . IFN-γ was used as a positive control, and WP1066 was used to inhibit STAT3. (D) HUV-EC-C were treated with SPRC (100 μ M ) for 45 min. The nuclear extract was isolated and used to perform chromatin immunoprecipitation with an STAT3 antibody. The change of downstream promoters was analyzed by real-time polymerase chain reaction. # p

Techniques Used: Translocation Assay, Activation Assay, Isolation, Western Blot, Staining, Electrophoretic Mobility Shift Assay, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

23) Product Images from "MicroRNA-124 Regulates STAT3 Expression and is Downregulated in Colon Tissues of Pediatric Patients with Ulcerative Colitis"

Article Title: MicroRNA-124 Regulates STAT3 Expression and is Downregulated in Colon Tissues of Pediatric Patients with Ulcerative Colitis

Journal: Gastroenterology

doi: 10.1053/j.gastro.2013.07.001

Inverse correlation of miR-124 and STAT3 levels is specific to pediatric biopsies of active UC. ( A ) Expression levels of miR-124 in pediatric non-IBD (pControl, n = 8), pediatric active UC (pUC, n = 7), pediatric Crohn’s disease (pCD, n = 6), adult non-IBD (aControl, n = 6) and adult UC (aUC, n = 11) patient biopsies, determined by real-time PCR. Results are presented as boxes with whiskers (minimum-to-maximum), relative to pediatric-control samples (pControl). ns: not statistically significant (One-way ANOVA, Prism6, GraphPad Software Inc.). ( B ) Correlation of miR-124 levels to STAT3 mRNA levels in pediatric-UC and pediatric-Control biopsies. Correlation coefficient was calculated using Prism6 (GraphPad Software, Inc.). ( C ) Expression levels of miR-124 in biopsies from pediatric patients with inactive UC (Inactive-pUC, n = 6), compared to active pediatric-UC biopsies (Active-pUC, n = 6). ( D ) Western blot analysis of lysates from frozen biopsies from inactive UC (lanes 1 to 5) and active UC (lanes 6 to 9) pediatric patients for p-STAT3 and quantitation of the intensity of the detected bands, normalized to GAPDH levels and expressed in arbitrary units (AU). Quantitation for each group (right panel); data are presented as mean ±SE of the mean. t -test analysis was performed using Prism6 (GraphPad Software, Inc.). ( E ) Immunostaining for p-STAT3 in biopsies from inactive and active pediatric-UC patients. Representative microscopy pictures are shown in original magnification 20× (insert) and 40× (magnified dotted area), as indicated. Scale bars represent 50 µm.
Figure Legend Snippet: Inverse correlation of miR-124 and STAT3 levels is specific to pediatric biopsies of active UC. ( A ) Expression levels of miR-124 in pediatric non-IBD (pControl, n = 8), pediatric active UC (pUC, n = 7), pediatric Crohn’s disease (pCD, n = 6), adult non-IBD (aControl, n = 6) and adult UC (aUC, n = 11) patient biopsies, determined by real-time PCR. Results are presented as boxes with whiskers (minimum-to-maximum), relative to pediatric-control samples (pControl). ns: not statistically significant (One-way ANOVA, Prism6, GraphPad Software Inc.). ( B ) Correlation of miR-124 levels to STAT3 mRNA levels in pediatric-UC and pediatric-Control biopsies. Correlation coefficient was calculated using Prism6 (GraphPad Software, Inc.). ( C ) Expression levels of miR-124 in biopsies from pediatric patients with inactive UC (Inactive-pUC, n = 6), compared to active pediatric-UC biopsies (Active-pUC, n = 6). ( D ) Western blot analysis of lysates from frozen biopsies from inactive UC (lanes 1 to 5) and active UC (lanes 6 to 9) pediatric patients for p-STAT3 and quantitation of the intensity of the detected bands, normalized to GAPDH levels and expressed in arbitrary units (AU). Quantitation for each group (right panel); data are presented as mean ±SE of the mean. t -test analysis was performed using Prism6 (GraphPad Software, Inc.). ( E ) Immunostaining for p-STAT3 in biopsies from inactive and active pediatric-UC patients. Representative microscopy pictures are shown in original magnification 20× (insert) and 40× (magnified dotted area), as indicated. Scale bars represent 50 µm.

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Software, Western Blot, Quantitation Assay, Immunostaining, Microscopy

24) Product Images from "Selective ?2-Adrenoreceptor Stimulation Attenuates Myocardial Cell Death and Preserves Cardiac Function After Ischemia-Reperfusion Injury"

Article Title: Selective ?2-Adrenoreceptor Stimulation Attenuates Myocardial Cell Death and Preserves Cardiac Function After Ischemia-Reperfusion Injury

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

doi: 10.1161/ATVBAHA.112.251769

A , Representative immunoblots of endothelial NO synthase (eNOS) total protein, eNOS P- Ser1177 , eNOS P Thr495 , and GAPDH in tissue samples taken from the left ventricle of sham-operated mice at 15 minutes after administration of either vehicle (VEH) or
Figure Legend Snippet: A , Representative immunoblots of endothelial NO synthase (eNOS) total protein, eNOS P- Ser1177 , eNOS P Thr495 , and GAPDH in tissue samples taken from the left ventricle of sham-operated mice at 15 minutes after administration of either vehicle (VEH) or

Techniques Used: Western Blot, Mouse Assay

25) Product Images from "Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor"

Article Title: Berberine Induces Caspase-Independent Cell Death in Colon Tumor Cells through Activation of Apoptosis-Inducing Factor

Journal: PLoS ONE

doi: 10.1371/journal.pone.0036418

Berberine stimulates AIF release from the mitochondria to the cytoplasm and nuclear translocation in IMCE, which is required for berberine-induced cell death. Cells were treated with berberine at 50 µM for the indicated times, as described in Figure 2 . Cytoplasmic, nuclear, and mitochondrial fractions were prepared for Western blot analysis using anti-AIF, anti-GAPDH (cytosolic marker), anti-COX IV (mitochondrial marker) and anti-Ki-67 (nuclear marker) antibodies (A). Immunostaining of cells with 50 µM berberine treatment for 18 h was performed to localize AIF using an anti-AIF antibody and a FITC-conjugated secondary antibody (green) and nuclei using DAPI staining (blue). Arrows indicate nuclei with AIF translocation (B). IMCE cells transfected with AIF siRNA or non-targeting siRNA for 24 h were treated with berberine at 50 µM for 18 h to detect number change (D) and LDH release (E) as described in Figure 2 . AIF expression levels were detected by Western blot analysis using an anti-AIF antibody (C). Actin blot was used as a protein loading control. * p
Figure Legend Snippet: Berberine stimulates AIF release from the mitochondria to the cytoplasm and nuclear translocation in IMCE, which is required for berberine-induced cell death. Cells were treated with berberine at 50 µM for the indicated times, as described in Figure 2 . Cytoplasmic, nuclear, and mitochondrial fractions were prepared for Western blot analysis using anti-AIF, anti-GAPDH (cytosolic marker), anti-COX IV (mitochondrial marker) and anti-Ki-67 (nuclear marker) antibodies (A). Immunostaining of cells with 50 µM berberine treatment for 18 h was performed to localize AIF using an anti-AIF antibody and a FITC-conjugated secondary antibody (green) and nuclei using DAPI staining (blue). Arrows indicate nuclei with AIF translocation (B). IMCE cells transfected with AIF siRNA or non-targeting siRNA for 24 h were treated with berberine at 50 µM for 18 h to detect number change (D) and LDH release (E) as described in Figure 2 . AIF expression levels were detected by Western blot analysis using an anti-AIF antibody (C). Actin blot was used as a protein loading control. * p

Techniques Used: Translocation Assay, Western Blot, Marker, Immunostaining, Staining, Transfection, Expressing

Berberine-stimulated cathepsin B release through ROS production is required for AIF activation in IMCE. Cells were treated with berberine at 50 µM for 18 h in the presence or absence of a ROS scavenger, Tiron (5 µM) or a cathepsin B inhibitor, CA-074 Me (100 nM). Immunostaining was performed to localize AIF using an anti-cathepsin B antibody and a FITC-conjugated secondary antibody (green) and nuclei using DAPI staining (blue) (A). Cathepsin B staining shows granular lysosomal pattern in control cells and berberine treated YAMC cells and a diffuse cytosolic pattern with more nuclear distribution after berberine treatment in IMCE cells. Cytoplasmic fractions were prepared for Western blot analysis using anti-cathepsin B, anti-AIF, anti-GAPDH antibodies (B). Cell number change (C) and LDH release (D) were detected as described in Figure 2 . * p
Figure Legend Snippet: Berberine-stimulated cathepsin B release through ROS production is required for AIF activation in IMCE. Cells were treated with berberine at 50 µM for 18 h in the presence or absence of a ROS scavenger, Tiron (5 µM) or a cathepsin B inhibitor, CA-074 Me (100 nM). Immunostaining was performed to localize AIF using an anti-cathepsin B antibody and a FITC-conjugated secondary antibody (green) and nuclei using DAPI staining (blue) (A). Cathepsin B staining shows granular lysosomal pattern in control cells and berberine treated YAMC cells and a diffuse cytosolic pattern with more nuclear distribution after berberine treatment in IMCE cells. Cytoplasmic fractions were prepared for Western blot analysis using anti-cathepsin B, anti-AIF, anti-GAPDH antibodies (B). Cell number change (C) and LDH release (D) were detected as described in Figure 2 . * p

Techniques Used: Activation Assay, Immunostaining, Staining, Western Blot

26) Product Images from "Ganoderiol A-Enriched Extract Suppresses Migration and Adhesion of MDA-MB-231 Cells by Inhibiting FAK-SRC-Paxillin Cascade Pathway"

Article Title: Ganoderiol A-Enriched Extract Suppresses Migration and Adhesion of MDA-MB-231 Cells by Inhibiting FAK-SRC-Paxillin Cascade Pathway

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076620

GAEE suppressed FAK signaling in MDA-MB-231 cells. (A) Immunoblots of FAK signaling proteins and other related proteins after 24 h of treatment with GAEE in MDA-MB-231 cells. Decreased expression of FAK, p-FAK (Y397), and p-FAK (Y925) were shown, while there is no alter on the expression of SRC, p-SRC, integrin β1, and integrin β4. (B) The relative densities of FAK, p-FAK (Y397), integrin β1 and integrin β4 were normalized against GAPDH by densitometric analysis. The values represented as the mean ± SEM of four independent experiments compared with control group. * P
Figure Legend Snippet: GAEE suppressed FAK signaling in MDA-MB-231 cells. (A) Immunoblots of FAK signaling proteins and other related proteins after 24 h of treatment with GAEE in MDA-MB-231 cells. Decreased expression of FAK, p-FAK (Y397), and p-FAK (Y925) were shown, while there is no alter on the expression of SRC, p-SRC, integrin β1, and integrin β4. (B) The relative densities of FAK, p-FAK (Y397), integrin β1 and integrin β4 were normalized against GAPDH by densitometric analysis. The values represented as the mean ± SEM of four independent experiments compared with control group. * P

Techniques Used: Multiple Displacement Amplification, Western Blot, Expressing

GAEE inhibited activation of paxillin in MDA-MB-231 cells. (A) Cells were treated with increasing concentrations of GAEE for 24 h, lysed, and immunoblotting with paxillin, p-paxillin (Y118). (B) The relative density of p-paxillin (Y118) was normalized against GAPDH by densitometric analysis. The values represented as the mean ±SEM of four independent experiments (* P
Figure Legend Snippet: GAEE inhibited activation of paxillin in MDA-MB-231 cells. (A) Cells were treated with increasing concentrations of GAEE for 24 h, lysed, and immunoblotting with paxillin, p-paxillin (Y118). (B) The relative density of p-paxillin (Y118) was normalized against GAPDH by densitometric analysis. The values represented as the mean ±SEM of four independent experiments (* P

Techniques Used: Activation Assay, Multiple Displacement Amplification

27) Product Images from "Lrf suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion"

Article Title: Lrf suppresses prostate cancer through repression of a Sox9-dependent pathway for cellular senescence bypass and tumor invasion

Journal: Nature genetics

doi: 10.1038/ng.2654

Loss of Lrf leads to senescence bypass and increased proliferation (a) Senescence-associated β-galactosidase staining (SA-β-gal) of WT , Lrf flox/flox ; Pb-Cre4 , Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates of 12 week-old mice show a significant reduction of senescence in Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates as compared to Pten flox/flox ;Pb-Cre4 prostates. (b) Percentage of SA-b-gal positive cells in the prostate of 12 week-old Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 mice (number of mice=3/genotype, number of cells=1000/field, number of fields=10/lobe). (c) anti-p53, anti-p27 and p19Arf staining in 12 week-old WT , Lrf flox/flox ; Pb-Cre4 , Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates. (d) Western blot analysis for Pten, pAkt (Serine 473), Lrf, Smad4, pSmad2, AR, p53, p21, p27, and p19Arf. (e) Ki-67 staining of WT , Lrf flox/flox ; Pb-Cre4 , Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates suggests that loss of Lrf and Pten leads to increased proliferation. (f) Percentage of Ki67 positive cells in the three lobes of WT (light grey bars), Lrf flox/flox ; Pb-Cre4 (grey bars), Pten flox/flox ;Pb-Cre4 (dark grey bars), and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 (black bars) 12-week-old mice (number of mice=3/genotype, total cells/lobe=5000). (g) Western blot analysis for cleaved caspase 3, total caspase 3, and Gapdh of Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 12-week-old mice prostates.
Figure Legend Snippet: Loss of Lrf leads to senescence bypass and increased proliferation (a) Senescence-associated β-galactosidase staining (SA-β-gal) of WT , Lrf flox/flox ; Pb-Cre4 , Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates of 12 week-old mice show a significant reduction of senescence in Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates as compared to Pten flox/flox ;Pb-Cre4 prostates. (b) Percentage of SA-b-gal positive cells in the prostate of 12 week-old Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 mice (number of mice=3/genotype, number of cells=1000/field, number of fields=10/lobe). (c) anti-p53, anti-p27 and p19Arf staining in 12 week-old WT , Lrf flox/flox ; Pb-Cre4 , Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates. (d) Western blot analysis for Pten, pAkt (Serine 473), Lrf, Smad4, pSmad2, AR, p53, p21, p27, and p19Arf. (e) Ki-67 staining of WT , Lrf flox/flox ; Pb-Cre4 , Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 prostates suggests that loss of Lrf and Pten leads to increased proliferation. (f) Percentage of Ki67 positive cells in the three lobes of WT (light grey bars), Lrf flox/flox ; Pb-Cre4 (grey bars), Pten flox/flox ;Pb-Cre4 (dark grey bars), and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 (black bars) 12-week-old mice (number of mice=3/genotype, total cells/lobe=5000). (g) Western blot analysis for cleaved caspase 3, total caspase 3, and Gapdh of Pten flox/flox ;Pb-Cre4 , and Pten flox/flox ;Lrf flox/flox ; Pb-Cre4 12-week-old mice prostates.

Techniques Used: Staining, Mouse Assay, Western Blot

28) Product Images from "Nucleolar and spindle associated protein 1 promotes metastasis of cervical carcinoma cells by activating Wnt/β-catenin signaling"

Article Title: Nucleolar and spindle associated protein 1 promotes metastasis of cervical carcinoma cells by activating Wnt/β-catenin signaling

Journal: Journal of Experimental & Clinical Cancer Research : CR

doi: 10.1186/s13046-019-1037-y

NUSAP1 is upregulated in cervical cancer. a NUSAP1 mRNA expression levels were significantly upregulated in cervical cancer tissues compared with normal tissues, as indicated by The Cancer Genome Atlas (COAD) cervical cancer dataset and public GEO cervical cancer datasets GSE7803 and GSE9750. d , e Real-time PCR analysis of NUSAP1 mRNA in ECT1/E6E7 human immortalized cervical epithelial cells and in eight cultured cervical cancer cell lines ( d ) and seven fresh primary cervical cancer tissues as compared with paired normal tissues ( e ). f Real-time PCR analysis of NUSAP1 mRNA in six cervical cancer samples without metastasis and eight cervical cancer samples with metastasis within 5 years; GAPDH was used as a loading control. g , h Western blotting analysis of NUSAP1 expression in ECT1/E6E7 human immortalized cervical epithelial cells and eight cultured cervical cancer cell lines ( g ), and in seven paired fresh primary cervical cancer tissues and adjacent non-cancerous tissue sections ( h ). i Western blotting results of NUSAP1 expression in six cervical cancer cases without metastasis and eight cervical cancer specimens with metastasis within 5 years; α-tubulin was used as a loading control. All experiments were performed in triplicate. Results are the mean ± SD. ** p
Figure Legend Snippet: NUSAP1 is upregulated in cervical cancer. a NUSAP1 mRNA expression levels were significantly upregulated in cervical cancer tissues compared with normal tissues, as indicated by The Cancer Genome Atlas (COAD) cervical cancer dataset and public GEO cervical cancer datasets GSE7803 and GSE9750. d , e Real-time PCR analysis of NUSAP1 mRNA in ECT1/E6E7 human immortalized cervical epithelial cells and in eight cultured cervical cancer cell lines ( d ) and seven fresh primary cervical cancer tissues as compared with paired normal tissues ( e ). f Real-time PCR analysis of NUSAP1 mRNA in six cervical cancer samples without metastasis and eight cervical cancer samples with metastasis within 5 years; GAPDH was used as a loading control. g , h Western blotting analysis of NUSAP1 expression in ECT1/E6E7 human immortalized cervical epithelial cells and eight cultured cervical cancer cell lines ( g ), and in seven paired fresh primary cervical cancer tissues and adjacent non-cancerous tissue sections ( h ). i Western blotting results of NUSAP1 expression in six cervical cancer cases without metastasis and eight cervical cancer specimens with metastasis within 5 years; α-tubulin was used as a loading control. All experiments were performed in triplicate. Results are the mean ± SD. ** p

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

29) Product Images from "The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages"

Article Title: The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006057

Detection of ORF m42-encoded proteins and kinetics of CD45 down-regulation. (A) Predicted amino acid sequence of the m42 protein with the LPTY (dotted line) and PPSY (underlined) motifs and the putative membrane anchor (gray) marked. (B) RAW264.7 cells were either mock-infected or infected with MCMVgfp, MCMVgfp-m42STOP or MCMVgfp-m42rev, and 24 h p.i. cells were analyzed by immunoblotting for CD45 and m42 expression. IE1 (immediate-early protein 1) served as infection marker and GAPDH as loading control. (C, D) RAW264.7 cells were infected with MCMVgfp, lysed at the indicated time points and expression of CD45 and the viral proteins m42, IE1, E1 and M57 was analyzed by immunoblotting with the respective antibodies. E1, early protein 1; M57, ssDNA-binding protein. (D) In parallel, CD45 surface expression in the MCMVgfp-infected RAW264.7 cells was assessed by flow cytometry. (E, left panel) CD45 surface levels of the parental RAW264.7 cells, the control cell line RAW_ctrl (obtained after transduction with the empty retroviral vector), and the m42-expressing cells were examined by flow cytometry. (right) Expression of CD45, m42 and GAPDH (loading control) in the respective cell lines was determined by immunoblotting.
Figure Legend Snippet: Detection of ORF m42-encoded proteins and kinetics of CD45 down-regulation. (A) Predicted amino acid sequence of the m42 protein with the LPTY (dotted line) and PPSY (underlined) motifs and the putative membrane anchor (gray) marked. (B) RAW264.7 cells were either mock-infected or infected with MCMVgfp, MCMVgfp-m42STOP or MCMVgfp-m42rev, and 24 h p.i. cells were analyzed by immunoblotting for CD45 and m42 expression. IE1 (immediate-early protein 1) served as infection marker and GAPDH as loading control. (C, D) RAW264.7 cells were infected with MCMVgfp, lysed at the indicated time points and expression of CD45 and the viral proteins m42, IE1, E1 and M57 was analyzed by immunoblotting with the respective antibodies. E1, early protein 1; M57, ssDNA-binding protein. (D) In parallel, CD45 surface expression in the MCMVgfp-infected RAW264.7 cells was assessed by flow cytometry. (E, left panel) CD45 surface levels of the parental RAW264.7 cells, the control cell line RAW_ctrl (obtained after transduction with the empty retroviral vector), and the m42-expressing cells were examined by flow cytometry. (right) Expression of CD45, m42 and GAPDH (loading control) in the respective cell lines was determined by immunoblotting.

Techniques Used: Sequencing, Infection, Expressing, Marker, Binding Assay, Flow Cytometry, Cytometry, Transduction, Plasmid Preparation

Unaffected CD45 expression in RAW264.7 cell lines expressing m42 variants with a mutated PPSY motif. (A) Analysis of CD45 and m42 levels in RAW264.7 cells stably expressing m42 variants with disrupted PPxY motif. Lysates of RAW264.7, RAW_ctrl, RAW_m42, RAW_m42AASY and RAW_m42PPSA cells were examined by immunoblotting for expression of CD45 and m42. GAPDH served as loading control. (B) CD45 and CD71 surface expression of the indicated cell line was measured by flow cytometry. (C) Proposed model for m42-mediated down-regulation of CD45. By interaction of m42 with Nedd4-like ubiquitin ligases of the HECT family, a conformational change is induced. The thereby activated ubiquitin ligase marks CD45 for internalization and subsequent degradation.
Figure Legend Snippet: Unaffected CD45 expression in RAW264.7 cell lines expressing m42 variants with a mutated PPSY motif. (A) Analysis of CD45 and m42 levels in RAW264.7 cells stably expressing m42 variants with disrupted PPxY motif. Lysates of RAW264.7, RAW_ctrl, RAW_m42, RAW_m42AASY and RAW_m42PPSA cells were examined by immunoblotting for expression of CD45 and m42. GAPDH served as loading control. (B) CD45 and CD71 surface expression of the indicated cell line was measured by flow cytometry. (C) Proposed model for m42-mediated down-regulation of CD45. By interaction of m42 with Nedd4-like ubiquitin ligases of the HECT family, a conformational change is induced. The thereby activated ubiquitin ligase marks CD45 for internalization and subsequent degradation.

Techniques Used: Expressing, Stable Transfection, Flow Cytometry, Cytometry

Inhibition of CD45 downregulation in m42-expressing cells by lysosomal inhibitors and interference of m42 with Nedd4-like E3 ubiquitin ligases. (A) RAW264.7 or RAW_m42 were left untreated or were treated with proteasome inhibitors (MG132, Epoxomycin) or lysosome inhibitors (NH 4 Cl, Bafilomycin A1, Leupeptin) for 4 h, followed by immunoblot analysis of CD45 and m42 expression. GAPDH served as loading control. (B, C) HEK 293T cells or a HEK 293T-based cell line stably expressing m42 were either mock transfected or transfected with an empty vector (EV) or a vector encoding murine CD45RB. 48 h post transfection cell lysates were analyzed by immunoblotting for CD45, m42, Itch, Smurf2 and Nedd4 protein expression. α-tubulin served as loading control. (D) HEK 293T cells were transfected with expression vectors for m42 or CD45RB or both vectors and 48 h later cell lysates were subjected to immunoprecipitation with a CD45 antibody. Proteins in lysates and precipitates were detected by immunoblotting using the indicated specific antibodies. LC indicates the signals of the antibody light chain.
Figure Legend Snippet: Inhibition of CD45 downregulation in m42-expressing cells by lysosomal inhibitors and interference of m42 with Nedd4-like E3 ubiquitin ligases. (A) RAW264.7 or RAW_m42 were left untreated or were treated with proteasome inhibitors (MG132, Epoxomycin) or lysosome inhibitors (NH 4 Cl, Bafilomycin A1, Leupeptin) for 4 h, followed by immunoblot analysis of CD45 and m42 expression. GAPDH served as loading control. (B, C) HEK 293T cells or a HEK 293T-based cell line stably expressing m42 were either mock transfected or transfected with an empty vector (EV) or a vector encoding murine CD45RB. 48 h post transfection cell lysates were analyzed by immunoblotting for CD45, m42, Itch, Smurf2 and Nedd4 protein expression. α-tubulin served as loading control. (D) HEK 293T cells were transfected with expression vectors for m42 or CD45RB or both vectors and 48 h later cell lysates were subjected to immunoprecipitation with a CD45 antibody. Proteins in lysates and precipitates were detected by immunoblotting using the indicated specific antibodies. LC indicates the signals of the antibody light chain.

Techniques Used: Inhibition, Expressing, Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation

30) Product Images from "A Novel Combination RNAi toward Warburg Effect by Replacement with miR-145 and Silencing of PTBP1 Induces Apoptotic Cell Death in Bladder Cancer Cells"

Article Title: A Novel Combination RNAi toward Warburg Effect by Replacement with miR-145 and Silencing of PTBP1 Induces Apoptotic Cell Death in Bladder Cancer Cells

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms18010179

Increased expression levels of polypyrimidine tract-binding protein 1 (PTBP1) and pyruvate kinase muscle isoform 2 (PKM2) were demonstrated in clinical tumor samples from bladder cancer patients. PTBP1 and PKM2 expression in 12 bladder cancer clinical samples as determined by Western blot analysis. Details of the characteristics of the samples are given in Table 1 . GAPDH was used as the control.
Figure Legend Snippet: Increased expression levels of polypyrimidine tract-binding protein 1 (PTBP1) and pyruvate kinase muscle isoform 2 (PKM2) were demonstrated in clinical tumor samples from bladder cancer patients. PTBP1 and PKM2 expression in 12 bladder cancer clinical samples as determined by Western blot analysis. Details of the characteristics of the samples are given in Table 1 . GAPDH was used as the control.

Techniques Used: Expressing, Binding Assay, Western Blot

31) Product Images from "Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages"

Article Title: Lysosomal lipid hydrolysis provides substrates for lipid mediator synthesis in murine macrophages

Journal: Oncotarget

doi: 10.18632/oncotarget.16673

Lysosomal accumulation of lipids in Lal-/- macrophages A . Immunofluorescence staining of Wt and Lal-/- macrophages using BODIPY493/503 as neutral lipid dye and Cathepsin D as lysosomal marker. Co-localization analysis was performed using Mander's coefficient ( n = 4-5) + SEM. B . mRNA and C . protein expression of the LD marker PLIN2, normalized to the expression of B . cyclophilin and C . GAPDH. Data are shown as means + SD ( n = 3-4). *** p ≤ 0.001.
Figure Legend Snippet: Lysosomal accumulation of lipids in Lal-/- macrophages A . Immunofluorescence staining of Wt and Lal-/- macrophages using BODIPY493/503 as neutral lipid dye and Cathepsin D as lysosomal marker. Co-localization analysis was performed using Mander's coefficient ( n = 4-5) + SEM. B . mRNA and C . protein expression of the LD marker PLIN2, normalized to the expression of B . cyclophilin and C . GAPDH. Data are shown as means + SD ( n = 3-4). *** p ≤ 0.001.

Techniques Used: Immunofluorescence, Staining, Marker, Expressing

Lal mRNA expression in Wt immune cells Total RNA of flow-sorted immune cells was isolated and Lal mRNA expression was analyzed by quantitative real-time PCR relative to the expression of Gapdh as housekeeping gene. Transcript expression levels are shown as fold change relative to mRNA levels in macrophages and represent means + SD ( n = 4).
Figure Legend Snippet: Lal mRNA expression in Wt immune cells Total RNA of flow-sorted immune cells was isolated and Lal mRNA expression was analyzed by quantitative real-time PCR relative to the expression of Gapdh as housekeeping gene. Transcript expression levels are shown as fold change relative to mRNA levels in macrophages and represent means + SD ( n = 4).

Techniques Used: Expressing, Flow Cytometry, Isolation, Real-time Polymerase Chain Reaction

32) Product Images from "?-Lapachone Ameliorates Lipotoxic Cardiomyopathy in Acyl CoA Synthase Transgenic Mice"

Article Title: ?-Lapachone Ameliorates Lipotoxic Cardiomyopathy in Acyl CoA Synthase Transgenic Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0091039

β-Lap stimulates the AMPK signaling pathway through activation of Sirt1. A . Mouse hearts lysates (1 mg) were immunoprecipitated with an anti-acetyl lysine antibody. The precipitates and whole extracts (50 µg) were analyzed by western blotting. The levels of the acetylated forms of LKB1, FOXO, p53, and PARP1 were estimated by measuring band densities using NIH Image J software. GAPDH was used as a loading control. B . Western blot analysis of the protein levels of ACS, p-AMPK, AMPK, p-ACC, and ACC. Mouse heart lysates (50 µg) were subjected to western blot analysis, and levels of phosphorylated AMPK and ACC were estimated by measuring band densities using NIH Image J software. GAPDH was used as a loading control. n = 3−4 per group. Significance was measured via two-way ANOVA. * p
Figure Legend Snippet: β-Lap stimulates the AMPK signaling pathway through activation of Sirt1. A . Mouse hearts lysates (1 mg) were immunoprecipitated with an anti-acetyl lysine antibody. The precipitates and whole extracts (50 µg) were analyzed by western blotting. The levels of the acetylated forms of LKB1, FOXO, p53, and PARP1 were estimated by measuring band densities using NIH Image J software. GAPDH was used as a loading control. B . Western blot analysis of the protein levels of ACS, p-AMPK, AMPK, p-ACC, and ACC. Mouse heart lysates (50 µg) were subjected to western blot analysis, and levels of phosphorylated AMPK and ACC were estimated by measuring band densities using NIH Image J software. GAPDH was used as a loading control. n = 3−4 per group. Significance was measured via two-way ANOVA. * p

Techniques Used: Activation Assay, Immunoprecipitation, Western Blot, Software

Knockdown of Sirt1 inhibits activation of the AMPK signaling pathway by β-Lap. A . H9C2 cells were transfected with a plasmid harboring a scrambled shRNA (Scr) or Sirt1 shRNA (sh-Sirt1). Cell extracts (50 µg) were used for western blot analysis of the Sirt1 protein level and band intensities were quantified using NIH Image J software. GAPDH was used as a loading control. B . H9C2 cells transfected with Scr or sh-Sirt1 were treated with vehicle (Veh) or β-lap (βL). Cell extracts (50 µg) were used for western blot analysis of the level of phosphorylated AMPK and ACC and band intensities were quantified using NIH Image J software. GAPDH was used as a loading control. n = 3−4 per group. Significance was measured via Student's t-test (panel A) and two-way ANOVA (panel B). * p
Figure Legend Snippet: Knockdown of Sirt1 inhibits activation of the AMPK signaling pathway by β-Lap. A . H9C2 cells were transfected with a plasmid harboring a scrambled shRNA (Scr) or Sirt1 shRNA (sh-Sirt1). Cell extracts (50 µg) were used for western blot analysis of the Sirt1 protein level and band intensities were quantified using NIH Image J software. GAPDH was used as a loading control. B . H9C2 cells transfected with Scr or sh-Sirt1 were treated with vehicle (Veh) or β-lap (βL). Cell extracts (50 µg) were used for western blot analysis of the level of phosphorylated AMPK and ACC and band intensities were quantified using NIH Image J software. GAPDH was used as a loading control. n = 3−4 per group. Significance was measured via Student's t-test (panel A) and two-way ANOVA (panel B). * p

Techniques Used: Activation Assay, Transfection, Plasmid Preparation, shRNA, Western Blot, Software

33) Product Images from "The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation"

Article Title: The transcription factor PU.1 promotes alternative macrophage polarization and asthmatic airway inflammation

Journal: Journal of Molecular Cell Biology

doi: 10.1093/jmcb/mjv042

IL-4 induces the expression of PU.1 and phosphorylation of STAT6. BMDMs from WT mice were cultured and stimulated with 5 ng/ml of IL-4 for 15, 30, and 60 min. ( A ) The phosphorylated STAT6, total STAT6, PU.1, and GAPDH were identified by western blot.
Figure Legend Snippet: IL-4 induces the expression of PU.1 and phosphorylation of STAT6. BMDMs from WT mice were cultured and stimulated with 5 ng/ml of IL-4 for 15, 30, and 60 min. ( A ) The phosphorylated STAT6, total STAT6, PU.1, and GAPDH were identified by western blot.

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

34) Product Images from "Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association"

Article Title: Recurrent viral infections associated with a homozygous CORO1A mutation that disrupts oligomerization and cytoskeletal association

Journal: The Journal of allergy and clinical immunology

doi: 10.1016/j.jaci.2015.08.020

Oligomerization and cytoskeletal distribution of the CORO1A S401fs mutant ( A ) Co-immunoprecipitation of WT and Mut CORO1A in lysates from HEK293T cells co-transfected with combinations of Myc- and FLAG-tagged WT and Mut-CORO1A and FLAG-tagged PYK2 as a negative control. Myc- and FLAG-IPs and total cell lysates were immunoblotted with anti-Myc antibody. ( B ) Immunoblot analysis of CORO1A in cytosolic and cytoskeletal fractions from EBV-B cells of normal control (NL) or patient (P1). GAPDH and PARP were immunoblotted as markers for cytosolic and cytoskeleton-associated proteins, respectively. ( C ) Quantification of CORO1A distribution in the cytosolic and cytoskeletal fractions. Columns and bars represent mean and standard error of two independent experiments, *p
Figure Legend Snippet: Oligomerization and cytoskeletal distribution of the CORO1A S401fs mutant ( A ) Co-immunoprecipitation of WT and Mut CORO1A in lysates from HEK293T cells co-transfected with combinations of Myc- and FLAG-tagged WT and Mut-CORO1A and FLAG-tagged PYK2 as a negative control. Myc- and FLAG-IPs and total cell lysates were immunoblotted with anti-Myc antibody. ( B ) Immunoblot analysis of CORO1A in cytosolic and cytoskeletal fractions from EBV-B cells of normal control (NL) or patient (P1). GAPDH and PARP were immunoblotted as markers for cytosolic and cytoskeleton-associated proteins, respectively. ( C ) Quantification of CORO1A distribution in the cytosolic and cytoskeletal fractions. Columns and bars represent mean and standard error of two independent experiments, *p

Techniques Used: Mutagenesis, Immunoprecipitation, Transfection, Negative Control

35) Product Images from "Overexpression of Sulf2 in idiopathic pulmonary fibrosis"

Article Title: Overexpression of Sulf2 in idiopathic pulmonary fibrosis

Journal: Glycobiology

doi: 10.1093/glycob/cwt010

Sulf2 knockdown alters the TGF-β1 response in A549 cells. (A) Phospho- and total-Smad2 and -Smad3 levels were analyzed by western blotting at 15 min to 2 h following TGF-β1 stimulation (left); quantification of P-Smad2/GAPDH, T-smad2/GAPDH
Figure Legend Snippet: Sulf2 knockdown alters the TGF-β1 response in A549 cells. (A) Phospho- and total-Smad2 and -Smad3 levels were analyzed by western blotting at 15 min to 2 h following TGF-β1 stimulation (left); quantification of P-Smad2/GAPDH, T-smad2/GAPDH

Techniques Used: Western Blot

36) Product Images from "Dual Role of the Trps1 Transcription Factor in Dentin Mineralization *"

Article Title: Dual Role of the Trps1 Transcription Factor in Dentin Mineralization *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M114.550129

Decreased expression of genes involved in phosphate homeostasis in Trps1-OE cells. A, qRT-PCR graphs depicting Phex , Vdr , and Fam20C mRNA expression during osteo-odontogenic differentiation in Trps1-OE cells in comparison with Cntr cells. Data are presented as the mean relative levels of Phex , Vdr , and Fam20C mRNA (normalized to β-actin) ±S.D. from three stable cell lines. Day 0 values for each cell line were arbitrarily set to 1 and used as a reference for remainder of days of differentiation. B, top panel, Western blots of Phex, Vdr, and Fam20C expression on day 6 and Phex expression on day 8 of differentiation on protein extracts isolated from Trps1-OE and Cntr cell lines. Bottom panel, results of densitometric quantification of the Western blot images. Protein levels were normalized to tubulin or Gapdh as depicted in the top panel. Asterisks denote statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.005).
Figure Legend Snippet: Decreased expression of genes involved in phosphate homeostasis in Trps1-OE cells. A, qRT-PCR graphs depicting Phex , Vdr , and Fam20C mRNA expression during osteo-odontogenic differentiation in Trps1-OE cells in comparison with Cntr cells. Data are presented as the mean relative levels of Phex , Vdr , and Fam20C mRNA (normalized to β-actin) ±S.D. from three stable cell lines. Day 0 values for each cell line were arbitrarily set to 1 and used as a reference for remainder of days of differentiation. B, top panel, Western blots of Phex, Vdr, and Fam20C expression on day 6 and Phex expression on day 8 of differentiation on protein extracts isolated from Trps1-OE and Cntr cell lines. Bottom panel, results of densitometric quantification of the Western blot images. Protein levels were normalized to tubulin or Gapdh as depicted in the top panel. Asterisks denote statistically significant differences (*, p ≤ 0.05; **, p ≤ 0.005).

Techniques Used: Expressing, Quantitative RT-PCR, Stable Transfection, Western Blot, Isolation

37) Product Images from "Vincamine prevents lipopolysaccharide induced inflammation and oxidative stress via thioredoxin reductase activation in human corneal epithelial cells"

Article Title: Vincamine prevents lipopolysaccharide induced inflammation and oxidative stress via thioredoxin reductase activation in human corneal epithelial cells

Journal: American Journal of Translational Research

doi:

Effects of vincamine on the expression levels of redox proteins. Effect of vincamine on the expression levels of TrxR, GR, Trx and GPx in LPS treated HCECs were analyzed by western blotting. Cells were treated with LPS and various concentrations of vincamine, then the levels of TrxR, GR, Trx and GPx were detected by Western blot. The relative expression of proteins was quantified densitometrically with the software ImageJ and calculated according to the reference bands of GAPDH.
Figure Legend Snippet: Effects of vincamine on the expression levels of redox proteins. Effect of vincamine on the expression levels of TrxR, GR, Trx and GPx in LPS treated HCECs were analyzed by western blotting. Cells were treated with LPS and various concentrations of vincamine, then the levels of TrxR, GR, Trx and GPx were detected by Western blot. The relative expression of proteins was quantified densitometrically with the software ImageJ and calculated according to the reference bands of GAPDH.

Techniques Used: Expressing, Western Blot, Software

38) Product Images from "Overcoming Resistance to the THZ Series of Covalent Transcriptional CDK Inhibitors"

Article Title: Overcoming Resistance to the THZ Series of Covalent Transcriptional CDK Inhibitors

Journal: Cell chemical biology

doi: 10.1016/j.chembiol.2017.11.007

ABC Transporter Upregulation Mediates THZ1 Resistance in NB and Lung Cancer Cells (A) Dose-responsecurvesforTHZ1 S and THZ1 R NBcellstreated with THZ1 for72 hr(left). Forall dose-response curves, fractionsofviablecells relativeto DMSO-treated cells are shown. Western blot (WB) analysis of the indicated proteins in THZ1 S and THZ1 R cells treated with DMSO or THZ1 (right) (Kelly, 1 μM; NGP, LAN5, SK-N-BE2 [BE2], 300 nM) for 6 hr. (B) qRT-PCR analysis ofABC transporter expression in THZ1 S and THZ1 R cells (left). Expression was normalized to levels in THZ1 S cells, with GAPDH used as an internal control. WB analysis of ABC proteins in THZ1 S versus THZ1 R cells (right). (C).Dose-response curves for THZ1 S versus THZ1 R lung cancer cells treated with THZ1 for 72 hr (left). WB analysis of the indicated proteins in THZ1 S and THZ1 R cells treated with DMSO or THZ1 (NCI-H82, H3122, 300 nM; PC9, 1 μM) for 6 hr (right). (D) qRT-PCR (left) and WB (right) analyses of ABCG2 in THZ1 S and THZ1 R cells. (E).Viability ofTHZ1 R Kelly NB cellstreated with THZ1 in combination with tariquidar at the indicated doses for72 hr (left). WB ofthe indicated proteins in THZ1 R Kelly cells treated with THZ1 at the indicated concentrations in combination with tariquidar (125 nM) for 6 hr (right). (F).Dose-response curves forTHZ1 S versusTHZ1 R H82 SCLC cells treated with THZ1 in combination with KO-143 (200 nM) ortariquidar (125 nM) for72 hr(left). Viability of THZ1 R PC9 cells treated with THZ1 in combination with the indicated doses of KO-143 for 72 hr (right). (G).Viability of THZ1 R Kelly cells expressing either a control shRNA or two individual shRNAs against ABCB1 and treated with THZ1 (left). WB ofthe indicated proteins in THZ1 R .
Figure Legend Snippet: ABC Transporter Upregulation Mediates THZ1 Resistance in NB and Lung Cancer Cells (A) Dose-responsecurvesforTHZ1 S and THZ1 R NBcellstreated with THZ1 for72 hr(left). Forall dose-response curves, fractionsofviablecells relativeto DMSO-treated cells are shown. Western blot (WB) analysis of the indicated proteins in THZ1 S and THZ1 R cells treated with DMSO or THZ1 (right) (Kelly, 1 μM; NGP, LAN5, SK-N-BE2 [BE2], 300 nM) for 6 hr. (B) qRT-PCR analysis ofABC transporter expression in THZ1 S and THZ1 R cells (left). Expression was normalized to levels in THZ1 S cells, with GAPDH used as an internal control. WB analysis of ABC proteins in THZ1 S versus THZ1 R cells (right). (C).Dose-response curves for THZ1 S versus THZ1 R lung cancer cells treated with THZ1 for 72 hr (left). WB analysis of the indicated proteins in THZ1 S and THZ1 R cells treated with DMSO or THZ1 (NCI-H82, H3122, 300 nM; PC9, 1 μM) for 6 hr (right). (D) qRT-PCR (left) and WB (right) analyses of ABCG2 in THZ1 S and THZ1 R cells. (E).Viability ofTHZ1 R Kelly NB cellstreated with THZ1 in combination with tariquidar at the indicated doses for72 hr (left). WB ofthe indicated proteins in THZ1 R Kelly cells treated with THZ1 at the indicated concentrations in combination with tariquidar (125 nM) for 6 hr (right). (F).Dose-response curves forTHZ1 S versusTHZ1 R H82 SCLC cells treated with THZ1 in combination with KO-143 (200 nM) ortariquidar (125 nM) for72 hr(left). Viability of THZ1 R PC9 cells treated with THZ1 in combination with the indicated doses of KO-143 for 72 hr (right). (G).Viability of THZ1 R Kelly cells expressing either a control shRNA or two individual shRNAs against ABCB1 and treated with THZ1 (left). WB ofthe indicated proteins in THZ1 R .

Techniques Used: Western Blot, Quantitative RT-PCR, Expressing, shRNA

39) Product Images from "MicroRNA-21 down-regulates inflammation and inhibits periodontitis"

Article Title: MicroRNA-21 down-regulates inflammation and inhibits periodontitis

Journal: Molecular immunology

doi: 10.1016/j.molimm.2018.05.008

MiR-21 ablation promotes P. gingivalis LPS-induced NF-κB activation. BMDM cells from miR-21 KO mice and their WT littermates were stimulated with P. gingivalis LPS (2 μg/ml). Cell lysates were collected at the time points (hours) indicated for Western blot analysis. (A) Blots were probed with the antibodies to IκB, phosphrylated NF-κBp65, and GAPDH as a loading control. Densitometric quantification of the ratio of (B) total- IκB, and (C) p-NF-κBp65 to GAPDH. Data represent mean ± S.E.M. from three experiments. Asterisks indicate statistically significant ( p
Figure Legend Snippet: MiR-21 ablation promotes P. gingivalis LPS-induced NF-κB activation. BMDM cells from miR-21 KO mice and their WT littermates were stimulated with P. gingivalis LPS (2 μg/ml). Cell lysates were collected at the time points (hours) indicated for Western blot analysis. (A) Blots were probed with the antibodies to IκB, phosphrylated NF-κBp65, and GAPDH as a loading control. Densitometric quantification of the ratio of (B) total- IκB, and (C) p-NF-κBp65 to GAPDH. Data represent mean ± S.E.M. from three experiments. Asterisks indicate statistically significant ( p

Techniques Used: Activation Assay, Mouse Assay, Western Blot

40) Product Images from "eIF5A2 regulates the resistance of gastric cancer cells to cisplatin via induction of EMT"

Article Title: eIF5A2 regulates the resistance of gastric cancer cells to cisplatin via induction of EMT

Journal: American Journal of Translational Research

doi:

EMT and correlation with sensitivity to cisplatin. A. Western blotting of the effect of cisplatin and/or Twist siRNA on Twist, E-cadherin, N-cadherin, vimentin and β-catenin protein expression in GC cell lines. GAPDH was used as the internal control. B. GC cell lines were treated with different concentrations of cisplatin (0, 3.125, 6.25, 12.5, 25, 50 μmol/L) and/or Twist siRNA (2 μg/ml). Cell viability was examined using the CCK-8 assay. C. Western blot was used to detect the interference effciency of Twist siRNA in GC cell lines. GAPDH was used as the internal control. D, E. GC cell lines were treated with cisplatin (0, 3.125, 6.25, 12.5, 25, 50 μmol/L) and/or Twist siRNA (2 μg/ml). Cell proliferation was examined using the EDU assay; the numbers of EDU positive blotting confirmed Twist siRNA effectively downregulated protein expression.
Figure Legend Snippet: EMT and correlation with sensitivity to cisplatin. A. Western blotting of the effect of cisplatin and/or Twist siRNA on Twist, E-cadherin, N-cadherin, vimentin and β-catenin protein expression in GC cell lines. GAPDH was used as the internal control. B. GC cell lines were treated with different concentrations of cisplatin (0, 3.125, 6.25, 12.5, 25, 50 μmol/L) and/or Twist siRNA (2 μg/ml). Cell viability was examined using the CCK-8 assay. C. Western blot was used to detect the interference effciency of Twist siRNA in GC cell lines. GAPDH was used as the internal control. D, E. GC cell lines were treated with cisplatin (0, 3.125, 6.25, 12.5, 25, 50 μmol/L) and/or Twist siRNA (2 μg/ml). Cell proliferation was examined using the EDU assay; the numbers of EDU positive blotting confirmed Twist siRNA effectively downregulated protein expression.

Techniques Used: Western Blot, Expressing, CCK-8 Assay, EdU Assay

eIF5A2 expression and correlation with EMT-related markers. A. Western blotting of the effect of si-eIF5A2 on eIF5A2, E-cadherin, N-cadherin,vimentin and β-catenin protein expression in GC cell lines. GAPDH was used as the internal control. B. Western blotting of the effect of cisplatin and/or si-eIF5A2 on eIF5A2, E-cadherin, N-cadherin,vimentin and β-catenin protein expression in GC cell lines. GAPDH was used as the internal control. C. Immunofluorescence assay of the effect of cisplatin and/or si-eIF5A2 on vimentin (Green) and E-cadherin (Red) protein expression in GC cell lines; nuclei were stained using DAPI (blue).
Figure Legend Snippet: eIF5A2 expression and correlation with EMT-related markers. A. Western blotting of the effect of si-eIF5A2 on eIF5A2, E-cadherin, N-cadherin,vimentin and β-catenin protein expression in GC cell lines. GAPDH was used as the internal control. B. Western blotting of the effect of cisplatin and/or si-eIF5A2 on eIF5A2, E-cadherin, N-cadherin,vimentin and β-catenin protein expression in GC cell lines. GAPDH was used as the internal control. C. Immunofluorescence assay of the effect of cisplatin and/or si-eIF5A2 on vimentin (Green) and E-cadherin (Red) protein expression in GC cell lines; nuclei were stained using DAPI (blue).

Techniques Used: Expressing, Western Blot, Immunofluorescence, Staining

Related Articles

Western Blot:

Article Title: Upregulation of Cleavage and Polyadenylation Specific Factor 4 in Lung Adenocarcinoma and Its Critical Role for Cancer Cell Survival and Proliferation
Article Snippet: .. Western blots were probed with antibodies against CPSF4 (Proteintech Group, Inc., Chicago, USA), phospho-PI3K p85 (Tyr458)/p55 (Tyr199), PI3K, phosphor-Akt (Ser473), Akt, pTyr202/Y204-ERK1/2, ERK1/2, pThr183/Tyr185-SAPK/JNK, SAPK/JNK, cleaved caspase-3, cleaved caspase-9 and GAPDH (Cell Signaling Technology, Beverly, MA).The protein bands were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). .. Lung adenocarcinomas tissue microarray Tissue microarray (diameter, 1.5 mm; depth, 4 µm) used for immunostaining analysis of CPSF4 protein expression was purchased from Shanghai Outdo Biotech (Shanghai, China, http://www.superchip.com.cn/ ).

Incubation:

Article Title: Pretreatment with LCK Inhibitors Chemosensitizes Cisplatin-Resistant Endometrioid Ovarian Tumors
Article Snippet: .. Membranes were incubated overnight at 4°C with primary antibodies against pLCK (Y394) (1:1000) (R & D Systems), GAPDH (1:1000) (Cell Signaling), and γ-H2AX (1:1000) (Cell Signaling). .. Secondary anti-mouse or anti-rabbit IgG antibodies conjugated to horse radish peroxidase (HRP) (1:3000) (Cell Signaling) or (1:25,000) (ProMega) were used.

Article Title: Administration of Pigment Epithelium-Derived Factor Inhibits Airway Inflammation and Remodeling in Chronic OVA-Induced Mice via VEGF Suppression
Article Snippet: .. The membrane was blocked for 1 hour with Tris-buffered saline containing 0.05% Tween 20 (TBST) plus 5% skim milk and then incubated with 1:1,000 anti-VEGF (Abcam, Cambridge, UK) or 1:1,000 anti-GAPDH (Cell Signaling Technology Inc., Beverly, MA, USA) overnight at 4℃. .. The membrane was washed 3 times with TBST and then incubated with HRP-conjugated goat antirabbit IgG for 1 hour at room temperature.

Fractionation:

Article Title: Endothelial and cardiomyocyte PI3Kβ divergently regulate cardiac remodelling in response to ischaemic injury
Article Snippet: .. 2.6 Immunoblotting and nuclear fractionation Immunoblotting and nuclear fractionation were performed as previously described., Histone H3 and GAPDH (Cell Signaling) were used as nuclear and cytosolic markers, respectively. .. 2.7 RNA sequencing and TaqMan RT-PCR RNA isolation and RNA sequencing were performed as described.

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    Cell Signaling Technology Inc anti gapdh antibody
    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on <t>JNK</t> phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; <t>GAPDH</t> antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p
    Anti Gapdh Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 172 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti gapdh antibody/product/Cell Signaling Technology Inc
    Average 94 stars, based on 172 article reviews
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    Cell Signaling Technology Inc gapdh
    p110β is expressed in CM nuclei and CM-p110β inactivation alters gene expression in the heart. ( A ) Nuclear fractionation analysis for p110β, <t>GAPDH,</t> and Histone <t>H3</t> in p110β-αMHC and p110βFlx left ventricular lysates and immunofluorescence images of p110β (green) and sarcomeric α actin (red) on isolated murine CMs. * P
    Gapdh, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 5163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gapdh/product/Cell Signaling Technology Inc
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    Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Journal: Neurochemistry International

    Article Title: Effect of Glutamate and Riluzole on Manganese-Induced Apoptotic Cell Signaling in Neuronally Differentiated Mouse P19 Cells

    doi: 10.1016/j.neuint.2012.04.015

    Figure Lengend Snippet: Western blots showing the effect of manganese, glutamate or riluzole or combination of the three on JNK phosphorylation in neuronally differentiated P19 cells. (A) Western blots showing the levels of phosphorylated JNK in differentiated P19 cells treated with the Mn (0.3 mM), glutamate (G; 5 mM) and /or riluzole (R; 10 μM) for 18 hrs.; GAPDH antibody was used as a loading control. (B) Graphical representation of band densities from four separate experiments. Results are presented as mean ± SE; Mn vs. control a - p

    Article Snippet: Phospho-JNK antibody, anti-GAPDH antibody and secondary antibody for Western blots were obtained from Cell Signaling (Danvers, MA) and Western lightning plus substrate to develop immunoblots was from Perkin Elmer (Waltham, MA).

    Techniques: Western Blot

    p110β is expressed in CM nuclei and CM-p110β inactivation alters gene expression in the heart. ( A ) Nuclear fractionation analysis for p110β, GAPDH, and Histone H3 in p110β-αMHC and p110βFlx left ventricular lysates and immunofluorescence images of p110β (green) and sarcomeric α actin (red) on isolated murine CMs. * P

    Journal: Cardiovascular Research

    Article Title: Endothelial and cardiomyocyte PI3Kβ divergently regulate cardiac remodelling in response to ischaemic injury

    doi: 10.1093/cvr/cvy298

    Figure Lengend Snippet: p110β is expressed in CM nuclei and CM-p110β inactivation alters gene expression in the heart. ( A ) Nuclear fractionation analysis for p110β, GAPDH, and Histone H3 in p110β-αMHC and p110βFlx left ventricular lysates and immunofluorescence images of p110β (green) and sarcomeric α actin (red) on isolated murine CMs. * P

    Article Snippet: 2.6 Immunoblotting and nuclear fractionation Immunoblotting and nuclear fractionation were performed as previously described., Histone H3 and GAPDH (Cell Signaling) were used as nuclear and cytosolic markers, respectively.

    Techniques: Expressing, Fractionation, Immunofluorescence, Isolation

    PTPN4 is the potential target of miR-181c-5p. Overexpression of miR-181c-5p results in reduced levels of protein expression (a) of PTPN4 in H9C2 cardiomyocytes. (b) mRNA and (c) protein expression in H9C2 cardiomyocytes with or without H/R stimulation. (d) mRNA and (e) protein expression in H9C2 cardiomyocytes with or without LPS stimulation. (f) mRNA and (g) protein expression in the postischemic myocardium of rat. Representative Western blots of PTPN4 and GAPDH in the miR-181c-5p agomir (h) or antagomir (i)-transfected H9C2 cardiomyocytes with or without H/R stimulation. Protein presence of PTPN4 was normalized to β -tubulin or GAPDH. mRNA levels are expressed as fold changes against the mRNA expression in H9C2 cardiomyocytes with no stimulation or myocardium in Sham group. Anti-NC: negative control of miR-181c-5p antagomir; data are shown as means ± SEM; ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: MiR-181c-5p Promotes Inflammatory Response during Hypoxia/Reoxygenation Injury by Downregulating Protein Tyrosine Phosphatase Nonreceptor Type 4 in H9C2 Cardiomyocytes

    doi: 10.1155/2020/7913418

    Figure Lengend Snippet: PTPN4 is the potential target of miR-181c-5p. Overexpression of miR-181c-5p results in reduced levels of protein expression (a) of PTPN4 in H9C2 cardiomyocytes. (b) mRNA and (c) protein expression in H9C2 cardiomyocytes with or without H/R stimulation. (d) mRNA and (e) protein expression in H9C2 cardiomyocytes with or without LPS stimulation. (f) mRNA and (g) protein expression in the postischemic myocardium of rat. Representative Western blots of PTPN4 and GAPDH in the miR-181c-5p agomir (h) or antagomir (i)-transfected H9C2 cardiomyocytes with or without H/R stimulation. Protein presence of PTPN4 was normalized to β -tubulin or GAPDH. mRNA levels are expressed as fold changes against the mRNA expression in H9C2 cardiomyocytes with no stimulation or myocardium in Sham group. Anti-NC: negative control of miR-181c-5p antagomir; data are shown as means ± SEM; ∗ P

    Article Snippet: Antibodies against Iκ Bα (1 : 1000), phospho-Iκ Bα (Ser32/36 ) (1 : 1000), p65 (1 : 1000), phospho-p65 (Ser536 ) (1 : 1000), GAPDH (1 : 1000), and β-tubulin (1 : 3000) were purchased from Cell Signaling Technology and used as primary antibodies.

    Techniques: Over Expression, Expressing, Western Blot, Transfection, Negative Control

    Reduction of PTPN4 mediated the proinflammatory effect of miR-181c-5p in H9C2 cardiomyocytes. (a) Transfection of cells with PTPN4 siRNA (PTPN4 KD ) resulted in significant reduction of PTPN4 protein level in H9C2 cardiomyocytes. (b) Representative Western blots of phosphorylated I κ B α (Ser 32/36 ), I κ B α , phosphorylated p65 (Ser 526 ), p65, and β -tubulin in the scramble siRNA or PTPN4 siRNA transfected H9C2 cardiomyocytes with or without H/R stimulation. (c) mRNA expression of NF κ B-dependent genes, including IL-1 β , IL-6, and TNF α in the scramble siRNA or PTPN4 siRNA-transfected H9C2 cardiomyocytes with H/R stimulation. mRNA levels are expressed as fold changes against those mRNA expressions in scramble siRNA-transfected H9C2 cardiomyocytes with no stimulation. (d) Representative Western blots of phosphorylated I κ B α (Ser 32/36 ), I κ B α , phosphorylated p65 (Ser 526 ), p65, and GAPDH in the H9C2 cardiomyocytes cotransfected with miR-181c-5p antagomir and PTPN4 siRNA and subjected to H/R stimulation. Protein presence of phosphorylated I κ B α (Ser 32/36 ), I κ B α , and phosphorylated p65 (Ser 526 ) was normalized to I κ B α , β -tubulin/GAPDH, and p65, respectively. Data are shown as means ± SEM; ∗ P

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: MiR-181c-5p Promotes Inflammatory Response during Hypoxia/Reoxygenation Injury by Downregulating Protein Tyrosine Phosphatase Nonreceptor Type 4 in H9C2 Cardiomyocytes

    doi: 10.1155/2020/7913418

    Figure Lengend Snippet: Reduction of PTPN4 mediated the proinflammatory effect of miR-181c-5p in H9C2 cardiomyocytes. (a) Transfection of cells with PTPN4 siRNA (PTPN4 KD ) resulted in significant reduction of PTPN4 protein level in H9C2 cardiomyocytes. (b) Representative Western blots of phosphorylated I κ B α (Ser 32/36 ), I κ B α , phosphorylated p65 (Ser 526 ), p65, and β -tubulin in the scramble siRNA or PTPN4 siRNA transfected H9C2 cardiomyocytes with or without H/R stimulation. (c) mRNA expression of NF κ B-dependent genes, including IL-1 β , IL-6, and TNF α in the scramble siRNA or PTPN4 siRNA-transfected H9C2 cardiomyocytes with H/R stimulation. mRNA levels are expressed as fold changes against those mRNA expressions in scramble siRNA-transfected H9C2 cardiomyocytes with no stimulation. (d) Representative Western blots of phosphorylated I κ B α (Ser 32/36 ), I κ B α , phosphorylated p65 (Ser 526 ), p65, and GAPDH in the H9C2 cardiomyocytes cotransfected with miR-181c-5p antagomir and PTPN4 siRNA and subjected to H/R stimulation. Protein presence of phosphorylated I κ B α (Ser 32/36 ), I κ B α , and phosphorylated p65 (Ser 526 ) was normalized to I κ B α , β -tubulin/GAPDH, and p65, respectively. Data are shown as means ± SEM; ∗ P

    Article Snippet: Antibodies against Iκ Bα (1 : 1000), phospho-Iκ Bα (Ser32/36 ) (1 : 1000), p65 (1 : 1000), phospho-p65 (Ser536 ) (1 : 1000), GAPDH (1 : 1000), and β-tubulin (1 : 3000) were purchased from Cell Signaling Technology and used as primary antibodies.

    Techniques: Transfection, Western Blot, Expressing

    Cisplatin resistant endometrioid cells treated with LCK inhibitors indicate decreased pLCK and ovarian endometrioid cells treated with LCK inhibitor indicate increased DNA double strand breaks. Protein lysates from cisplatin resistant ovarian endometrioid cancer cells (CP70) treated with vehicle (DMSO), LCK inhibitor saracatinib (Sar), or PP2 were immunoblotted for pLCK, with GAPDH used as a loading control. Values normalized to vehicle control. (A) Protein lysates from ovarian endometrioid cancer cells (TOV112D) treated with varied doses of saracatinib (Sar) were immunoblotted for γ H2AX, with GAPDH used as a loading control (B) .

    Journal: bioRxiv

    Article Title: Pretreatment with LCK Inhibitors Chemosensitizes Cisplatin-Resistant Endometrioid Ovarian Tumors

    doi: 10.1101/2020.06.18.154732

    Figure Lengend Snippet: Cisplatin resistant endometrioid cells treated with LCK inhibitors indicate decreased pLCK and ovarian endometrioid cells treated with LCK inhibitor indicate increased DNA double strand breaks. Protein lysates from cisplatin resistant ovarian endometrioid cancer cells (CP70) treated with vehicle (DMSO), LCK inhibitor saracatinib (Sar), or PP2 were immunoblotted for pLCK, with GAPDH used as a loading control. Values normalized to vehicle control. (A) Protein lysates from ovarian endometrioid cancer cells (TOV112D) treated with varied doses of saracatinib (Sar) were immunoblotted for γ H2AX, with GAPDH used as a loading control (B) .

    Article Snippet: Membranes were incubated overnight at 4°C with primary antibodies against pLCK (Y394) (1:1000) (R & D Systems), GAPDH (1:1000) (Cell Signaling), and γ-H2AX (1:1000) (Cell Signaling).

    Techniques: