mouse anti β actin (Millipore)

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Millipore mouse anti β actin

The resistance of CSP to IFN-γ-mediated killing of EEFs through the enhance of ATGs ubiquitination. ( A ) The mRNA levels of ATGs between control and CSP-stable transfected HepG2 cells in the heatmap were compared, and three biological repeats were performed. ( B ) The mRNA levels of ATGs (ATG3, ATG5, ATG7, and LC3) between control and CSP-stable transfected HepG2 cells were compared using real-time PCR. ( C ) Control and CSP-stable transfected HepG2 cells were treated with CHX for the indicated times, and the protein levels of LC3I/LC3II, ATG3, ATG5 and ATG7 were detected by western blot ( left ); the relative expression levels of LC3I/LC3II, ATG3, ATG5, and ATG7 to <t>β-actin</t> were quantified ( right ). ( D ) Control and CSP-stable transfected HepG2 cells were treated with the proteasome inhibitor MG132 for the indicated times, and the protein levels of LC3I/LC3II, ATG3, ATG5, and ATG7 were detected by western blot. ( E ) Control and CSP-stable transfected HepG2 cells were transfected with His-UB, and FLAG-ATG5 or FLAG-ATG7, and the ubiquitin binding to FLAG-ATG5 ( left ) or FLAG-ATG7 ( right ) in cells was detected by co-immunoprecipitation. UB, ubiquitin. ( F ) The levels of protein LC3, ATG5 and ATG7 were determined by western blot in both control and CSP-stable transfected HepG2 treated with or without 1μM TAK (TAK-243) for 24 h. ( G ) 1.2 × 10 5 CSP-stable transfected HepG2 or control cells were treated with or without 1μM TAK (TAK-243), and then were treated with IFN-γ and infected with 4 × 10 4 sporozoites for 46 h. The EEF number ( left ) and parasite load ( right ) were determined and compared as above. (H) IFN-γ R1 knockout and WT mice were per-treated with or without 20mg/kg TAK-243, and infected with 1,000 sporozoites in the next day, then liver parasite burden was determined at 46 h after infection as described as above. Data are represented as mean ± SEM; ns, not significant; * p

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1) Product Images from "ATGs ubiquitination is required for circumsporozoite protein to subvert host innate immunity against malaria liver stage"

Article Title: ATGs ubiquitination is required for circumsporozoite protein to subvert host innate immunity against malaria liver stage

Journal: bioRxiv

doi: 10.1101/2021.01.20.427456

Figure Legend Snippet: The resistance of CSP to IFN-γ-mediated killing of EEFs through the enhance of ATGs ubiquitination. ( A ) The mRNA levels of ATGs between control and CSP-stable transfected HepG2 cells in the heatmap were compared, and three biological repeats were performed. ( B ) The mRNA levels of ATGs (ATG3, ATG5, ATG7, and LC3) between control and CSP-stable transfected HepG2 cells were compared using real-time PCR. ( C ) Control and CSP-stable transfected HepG2 cells were treated with CHX for the indicated times, and the protein levels of LC3I/LC3II, ATG3, ATG5 and ATG7 were detected by western blot ( left ); the relative expression levels of LC3I/LC3II, ATG3, ATG5, and ATG7 to β-actin were quantified ( right ). ( D ) Control and CSP-stable transfected HepG2 cells were treated with the proteasome inhibitor MG132 for the indicated times, and the protein levels of LC3I/LC3II, ATG3, ATG5, and ATG7 were detected by western blot. ( E ) Control and CSP-stable transfected HepG2 cells were transfected with His-UB, and FLAG-ATG5 or FLAG-ATG7, and the ubiquitin binding to FLAG-ATG5 ( left ) or FLAG-ATG7 ( right ) in cells was detected by co-immunoprecipitation. UB, ubiquitin. ( F ) The levels of protein LC3, ATG5 and ATG7 were determined by western blot in both control and CSP-stable transfected HepG2 treated with or without 1μM TAK (TAK-243) for 24 h. ( G ) 1.2 × 10 5 CSP-stable transfected HepG2 or control cells were treated with or without 1μM TAK (TAK-243), and then were treated with IFN-γ and infected with 4 × 10 4 sporozoites for 46 h. The EEF number ( left ) and parasite load ( right ) were determined and compared as above. (H) IFN-γ R1 knockout and WT mice were per-treated with or without 20mg/kg TAK-243, and infected with 1,000 sporozoites in the next day, then liver parasite burden was determined at 46 h after infection as described as above. Data are represented as mean ± SEM; ns, not significant; * p

Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Binding Assay, Immunoprecipitation, Infection, Knock-Out, Mouse Assay

2) Product Images from "Combination therapy targeting the elevated interleukin‐6 level reduces invasive migration of BRAF inhibitor‐resistant melanoma cells"

Article Title: Combination therapy targeting the elevated interleukin‐6 level reduces invasive migration of BRAF inhibitor‐resistant melanoma cells

Journal: Molecular Oncology

doi: 10.1002/1878-0261.12433

Increased IL ‐6 secretion and WNT 5A expression are independent events in BRAF i‐R melanoma cells. Western blot analyses showing WNT 5A protein expression in IL ‐6 Ab‐treated HTB 63‐R (A) and A375‐R (B) melanoma cells. The graphs represent densitometric analyses of WNT 5A expression normalised against β‐actin from six separate experiments. The results are presented as WNT 5A expression in relation to that of nonresistant cells ( n = 6) and given as the mean ± SEM. Statistical analyses were performed using paired Student's t ‐tests. (C, D) IL ‐6 secretion from Box5‐treated HTB 63‐R and A375‐R melanoma cells was evaluated by ELISA as described in Materials and Methods . The ELISA analyses show the secreted IL ‐6 levels from Box5‐treated HTB 63‐R (C) and A375‐R (D) cells ( n = 6). The results on IL ‐6 secretion are presented in relation to that of nonresistant cells and given as the mean ± SEM. Statistical analyses were performed using paired Student's t ‐tests. (E, F) Cartoons outlining the IL ‐6/ WNT 5A positive feedback loop in (E) BRAF i‐sensitive melanoma cells and the independent IL ‐6 and WNT 5A signalling in (F) BRAF i‐R melanoma cells and how they affect invasive migration in these cells.

Figure Legend Snippet: Increased IL ‐6 secretion and WNT 5A expression are independent events in BRAF i‐R melanoma cells. Western blot analyses showing WNT 5A protein expression in IL ‐6 Ab‐treated HTB 63‐R (A) and A375‐R (B) melanoma cells. The graphs represent densitometric analyses of WNT 5A expression normalised against β‐actin from six separate experiments. The results are presented as WNT 5A expression in relation to that of nonresistant cells ( n = 6) and given as the mean ± SEM. Statistical analyses were performed using paired Student's t ‐tests. (C, D) IL ‐6 secretion from Box5‐treated HTB 63‐R and A375‐R melanoma cells was evaluated by ELISA as described in Materials and Methods . The ELISA analyses show the secreted IL ‐6 levels from Box5‐treated HTB 63‐R (C) and A375‐R (D) cells ( n = 6). The results on IL ‐6 secretion are presented in relation to that of nonresistant cells and given as the mean ± SEM. Statistical analyses were performed using paired Student's t ‐tests. (E, F) Cartoons outlining the IL ‐6/ WNT 5A positive feedback loop in (E) BRAF i‐sensitive melanoma cells and the independent IL ‐6 and WNT 5A signalling in (F) BRAF i‐R melanoma cells and how they affect invasive migration in these cells.

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

Development of BRAF i resistance in melanoma cells results in a significant increase in IL ‐6 secretion. (A) HTB 63 ( )/ HTB 63‐R ( ) and (B) A375 ( )/A375‐R ( ) cells were exposed to increasing concentrations of PLX 4032. MTT assays were performed as described in the methods section, and graphs were plotted using values from four independent experiments. The results are shown as the mean ± SEM. (C, D) Western blot analyses show the levels of ERK 1/2 activity (p‐ ERK 1/2 protein expression) in PLX 4032‐treated parental ( HTB 63 and A375) and PLX 4032‐resistant ( HTB 63‐R and A375‐R) melanoma cells. β‐Actin was used as a loading control in these experiments. Representative blots from four separate experiments are shown. The graphs represent the densitometric analyses of the ratio between p‐ ERK 1/2 and ERK 1/2 and show p‐ ERK 1/2 protein expression relative to that of vehicle‐treated nonresistant cells. The results are shown as the mean ± SEM. Statistical analyses were performed using both paired and unpaired Student's t ‐tests; * P

Figure Legend Snippet: Development of BRAF i resistance in melanoma cells results in a significant increase in IL ‐6 secretion. (A) HTB 63 ( )/ HTB 63‐R ( ) and (B) A375 ( )/A375‐R ( ) cells were exposed to increasing concentrations of PLX 4032. MTT assays were performed as described in the methods section, and graphs were plotted using values from four independent experiments. The results are shown as the mean ± SEM. (C, D) Western blot analyses show the levels of ERK 1/2 activity (p‐ ERK 1/2 protein expression) in PLX 4032‐treated parental ( HTB 63 and A375) and PLX 4032‐resistant ( HTB 63‐R and A375‐R) melanoma cells. β‐Actin was used as a loading control in these experiments. Representative blots from four separate experiments are shown. The graphs represent the densitometric analyses of the ratio between p‐ ERK 1/2 and ERK 1/2 and show p‐ ERK 1/2 protein expression relative to that of vehicle‐treated nonresistant cells. The results are shown as the mean ± SEM. Statistical analyses were performed using both paired and unpaired Student's t ‐tests; * P

Techniques Used: MTT Assay, Western Blot, Activity Assay, Expressing

Simultaneous inhibition of IL ‐6 and WNT 5A signalling reduces the invasive migration of BRAF i‐R melanoma cells through the inhibition of Cdc42‐ GTP ase activity. First, active Cdc42 levels were determined in parental and resistant HTB 63 (A) and A375 (B) cells through a pull‐down assay using GST ‐ PAK 1‐ PBD beads as described in the methods. The blots outline representative experiments showing the levels of Cdc42‐ GTP in the pull‐down and the total amounts of Cdc42 and β‐actin in the cell lysates. The graphs represent the densitometric analyses of the Cdc42‐ GTP protein bands in resistant cells relative to parental cells. Next, HTB 63‐R (C) and A375‐R (D) cells were treated with the combination of an IL ‐6 Ab and Box5, and Cdc42‐ GTP levels were analysed through pull‐down assays as mentioned in Materials and Methods . The blots outline representative experiments showing the levels of Cdc42‐ GTP that had been pulled down and the total amounts of Cdc42 and β‐actin in the cell lysates. The graphs represent densitometric analyses of the Cdc42‐ GTP protein band in IL ‐6 Ab and Box5‐treated cells relative to cells treated with vehicle. (E, F) HTB 63‐R cells were pretreated with vehicle or ML 141 for 48 h, the cells were washed and transferred to the inserts of the transwell migration or invasion plates, and the assays were performed for 24 h as described in Materials and Methods . The effects of the ML 141 treatment on migration and invasion are shown relative to that of vehicle‐treated ( DMSO ) HTB 63‐R cells. All the results in this figure are derived from separate experiments and are shown as the mean ± S.E.M. Statistical analyses were performed using (A, B) unpaired and (C–F) paired Student's t ‐tests; * P

Figure Legend Snippet: Simultaneous inhibition of IL ‐6 and WNT 5A signalling reduces the invasive migration of BRAF i‐R melanoma cells through the inhibition of Cdc42‐ GTP ase activity. First, active Cdc42 levels were determined in parental and resistant HTB 63 (A) and A375 (B) cells through a pull‐down assay using GST ‐ PAK 1‐ PBD beads as described in the methods. The blots outline representative experiments showing the levels of Cdc42‐ GTP in the pull‐down and the total amounts of Cdc42 and β‐actin in the cell lysates. The graphs represent the densitometric analyses of the Cdc42‐ GTP protein bands in resistant cells relative to parental cells. Next, HTB 63‐R (C) and A375‐R (D) cells were treated with the combination of an IL ‐6 Ab and Box5, and Cdc42‐ GTP levels were analysed through pull‐down assays as mentioned in Materials and Methods . The blots outline representative experiments showing the levels of Cdc42‐ GTP that had been pulled down and the total amounts of Cdc42 and β‐actin in the cell lysates. The graphs represent densitometric analyses of the Cdc42‐ GTP protein band in IL ‐6 Ab and Box5‐treated cells relative to cells treated with vehicle. (E, F) HTB 63‐R cells were pretreated with vehicle or ML 141 for 48 h, the cells were washed and transferred to the inserts of the transwell migration or invasion plates, and the assays were performed for 24 h as described in Materials and Methods . The effects of the ML 141 treatment on migration and invasion are shown relative to that of vehicle‐treated ( DMSO ) HTB 63‐R cells. All the results in this figure are derived from separate experiments and are shown as the mean ± S.E.M. Statistical analyses were performed using (A, B) unpaired and (C–F) paired Student's t ‐tests; * P

Techniques Used: Inhibition, Migration, Activity Assay, Pull Down Assay, Derivative Assay

3) Product Images from "Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2"

Article Title: Hepatitis B virus X protein promotes hepatoma cell proliferation via upregulation of MEKK2

Journal: Acta Pharmacologica Sinica

doi: 10.1038/aps.2011.52

HBx upregulates the expression of MEKK2. (A) The levels of MEKK2 and HBx were detected using RT-PCR and immunoblot analysis. GAPDH and β-actin were used as internal controls. (B) The levels of MEKK2 and HBx were detected in HepG2.2.15 cells using

Figure Legend Snippet: HBx upregulates the expression of MEKK2. (A) The levels of MEKK2 and HBx were detected using RT-PCR and immunoblot analysis. GAPDH and β-actin were used as internal controls. (B) The levels of MEKK2 and HBx were detected in HepG2.2.15 cells using

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction

4) Product Images from "Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells"

Article Title: Wnt signal activation induces midbrain specification through direct binding of the beta-catenin/TCF4 complex to the EN1 promoter in human pluripotent stem cells

Journal: Experimental & Molecular Medicine

doi: 10.1038/s12276-018-0044-y

Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

Figure Legend Snippet: Activation of Wnt signal induces midbrain characteristics in human ESC-derived NPCs. a Efficient induction of neural rosette cells from human ESCs by co-treatment with dorsomorphin and SB431542. b Strong immunoreactivity for SOX1 and Nestin in neural rosette cells. Morphology of neural rosette cells expanding in either the absence ( c ) or the presence of 1 μM BIO ( d ). e NPCs treated with BIO maintained immunoreactivity for SOX1 and Nestin. f Treatment with BIO upregulated EN1 expression and downregulated expressions of BF1 and GBX2 in dose-dependent manner. g BIO treatment significantly increased the number of EN1-positive neural cells. h The inductive effect of BIO treatment on midbrain fate appeared to be more specific than that of FGF8. i Expression pattern of another set of regional markers ( SIX3 for forebrain; PAX2 for midbrain; and HOXA2 for hindbrain) supported the midbrain-biased fate of NPCs treated with BIO. Treatment with other known GSK3 inhibitors, 1-AKP ( j ) and LiCl ( k ), resulted in regionalization comparable to BIO treatment. l Treating NPCs with Wnt antagonists (100 ng/ml DKK-1 and 500 ng/ml frizzled-5) downregulated the endogenous level of EN1 transcript in NPCs. m Immunoblot for β-catenin and EN1 protein after introduction of two different β-catenin-specific shRNAs (shRNA-1 and shRNA-2). EN1 protein level was directly downregulated by β-catenin knockdown. β-actin was a loading control. All data are expressed as mean ± S.E.M. Statistical significance was estimated using Student’s t test ( g , i , j , and k ) or one-way ANOVA ( f , h , and l ) from at least three independent experiments; * p

Techniques Used: Activation Assay, Derivative Assay, Expressing, ALP Assay, shRNA

5) Product Images from "SK-216, a Novel Inhibitor of Plasminogen Activator Inhibitor-1, Suppresses Lung Metastasis of Human Osteosarcoma"

Article Title: SK-216, a Novel Inhibitor of Plasminogen Activator Inhibitor-1, Suppresses Lung Metastasis of Human Osteosarcoma

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19030736

The PAI-1 inhibitor SK-216 suppresses invasion with no influence on proliferation or migration of 143B cells. ( a ) Western blot analyses of PAI-1 expression in SK-216 treated 143B cells. PAI-1 expression was quantified using Image Studio Lite (LI-COR) and normalized to β-actin. Expression is shown relative to that in non-treated cells (0 μM); ( b ) Matrigel assay of the invasion of SK-216-treated cells. The ratio of the number of pores containing invading cells to the total number of all pores is shown. Bar graphs show means ± SD ** p

Figure Legend Snippet: The PAI-1 inhibitor SK-216 suppresses invasion with no influence on proliferation or migration of 143B cells. ( a ) Western blot analyses of PAI-1 expression in SK-216 treated 143B cells. PAI-1 expression was quantified using Image Studio Lite (LI-COR) and normalized to β-actin. Expression is shown relative to that in non-treated cells (0 μM); ( b ) Matrigel assay of the invasion of SK-216-treated cells. The ratio of the number of pores containing invading cells to the total number of all pores is shown. Bar graphs show means ± SD ** p

Techniques Used: Migration, Western Blot, Expressing, Matrigel Assay

SK-216 suppresses PAI-1 expression of osteosarcoma cells in primary tumors. ( a ) Western blot analysis of PAI-1 expression (arrow) in primary tumors at 2 weeks after cell inoculation; ( b ) Western blot analysis of PAI-1 expression in 143B cells, which were transfected with PAI-1 siRNA or Control siRNA. Open circles and closed circles indicate upper and lower bands recognized by the anti-PAI-1 antibody used in this study, respectively; ( c ) PAI-1 expression in primary tumors was quantified using Image Studio Lite (LI-COR) and normalized to β-actin (±SD, n = 3 per group. * p

Figure Legend Snippet: SK-216 suppresses PAI-1 expression of osteosarcoma cells in primary tumors. ( a ) Western blot analysis of PAI-1 expression (arrow) in primary tumors at 2 weeks after cell inoculation; ( b ) Western blot analysis of PAI-1 expression in 143B cells, which were transfected with PAI-1 siRNA or Control siRNA. Open circles and closed circles indicate upper and lower bands recognized by the anti-PAI-1 antibody used in this study, respectively; ( c ) PAI-1 expression in primary tumors was quantified using Image Studio Lite (LI-COR) and normalized to β-actin (±SD, n = 3 per group. * p

Techniques Used: Expressing, Western Blot, Transfection

6) Product Images from "TNF?-mediated Hsd11b1 binding of NF-?B p65 is associated with suppression of 11?-HSD1 in muscle"

Article Title: TNF?-mediated Hsd11b1 binding of NF-?B p65 is associated with suppression of 11?-HSD1 in muscle

Journal: The Journal of Endocrinology

doi: 10.1530/JOE-13-0494

TNFα suppresses 11β-HSD1 mRNA and protein expression. (A) Real-time PCR analysis of 11β-HSD1 and H6PDH mRNA in C2C12 myotubes treated for 24 h with TNFα. (B) Western immunoblot analysis of p65 and β-actin from C2C12 myotubes treated for 24 h with TNFα compared to control. Densitometry of the western immunoblots were carried out using ImageJ (* P

Figure Legend Snippet: TNFα suppresses 11β-HSD1 mRNA and protein expression. (A) Real-time PCR analysis of 11β-HSD1 and H6PDH mRNA in C2C12 myotubes treated for 24 h with TNFα. (B) Western immunoblot analysis of p65 and β-actin from C2C12 myotubes treated for 24 h with TNFα compared to control. Densitometry of the western immunoblots were carried out using ImageJ (* P

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

7) Product Images from "Decline in DJ-1 and Decreased Nuclear Translocation of Nrf2 in Fuchs Endothelial Corneal Dystrophy"

Article Title: Decline in DJ-1 and Decreased Nuclear Translocation of Nrf2 in Fuchs Endothelial Corneal Dystrophy

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.12-10119

Effect of DJ-1 downregulation on Nrf2 localization. ( A ) Western blot analysis of DJ-1 levels in HCECi cells transfected with DJ-1 siRNA and scrambled siRNA. β-Actin was used for normalization of protein loading. ( B ) Representative fluorescence

Figure Legend Snippet: Effect of DJ-1 downregulation on Nrf2 localization. ( A ) Western blot analysis of DJ-1 levels in HCECi cells transfected with DJ-1 siRNA and scrambled siRNA. β-Actin was used for normalization of protein loading. ( B ) Representative fluorescence

Techniques Used: Western Blot, Transfection, Fluorescence

Decreased protein level of DJ-1. Left : representative bands of Western blot analysis of normal CECs and age- and sex-matched FECD specimens. β-Actin was used for normalization of protein loading. Jurkat cell lysate was used as a positive control.

Figure Legend Snippet: Decreased protein level of DJ-1. Left : representative bands of Western blot analysis of normal CECs and age- and sex-matched FECD specimens. β-Actin was used for normalization of protein loading. Jurkat cell lysate was used as a positive control.

Techniques Used: Western Blot, Positive Control

8) Product Images from "HTT silencing delays onset and slows progression of Huntington’s disease like phenotype: Monitoring with a novel neurovascular biomarker"

Article Title: HTT silencing delays onset and slows progression of Huntington’s disease like phenotype: Monitoring with a novel neurovascular biomarker

Journal: bioRxiv

doi: 10.1101/2020.11.17.386631

CRISPR/Cas9-mediated HTT silencing in the striatal neurons restores altered CBVa in the striatum of zQ175 mice. ( a ) Schematics of the designed HTT-gRNA (T1 and T3) and AAV vectors. ITR, inverted terminal repeat; HA, human influenza hemagglutinin; NLS, nuclear localization sequence; KASH, Klarsicht, ANC-1, Syne Homology; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; RFP, red fluorescent protein. ( b ) Timeline for AAV injections, HTT level measures and longitudinal CBVa assessment. ( c ) RFP fluorescence showing the transduction of AAV-HTT-gRNAs in the striatum. Scale bar = 900 μm (top image) and 40 μm (bottom image). ( c ) The images of Western blotting with indicated antibodies to HTT, NeuN, and β-actin. ( e ) Quantification results of mHTT (MW1), total HTT (combining upper and lower bands in the MCA2050 + MCA2051 blot), wtHTT (lower and major band in the blot with MAB 2166 antibody), and NeuN in the striatum injected with AAV-HTT gRNA + Cas9 (HTT gRNA, right striatum-R) or AAV-control gRNA + Cas9 (Con gRNA, left striatum-L). n=3, * p

Figure Legend Snippet: CRISPR/Cas9-mediated HTT silencing in the striatal neurons restores altered CBVa in the striatum of zQ175 mice. ( a ) Schematics of the designed HTT-gRNA (T1 and T3) and AAV vectors. ITR, inverted terminal repeat; HA, human influenza hemagglutinin; NLS, nuclear localization sequence; KASH, Klarsicht, ANC-1, Syne Homology; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; RFP, red fluorescent protein. ( b ) Timeline for AAV injections, HTT level measures and longitudinal CBVa assessment. ( c ) RFP fluorescence showing the transduction of AAV-HTT-gRNAs in the striatum. Scale bar = 900 μm (top image) and 40 μm (bottom image). ( c ) The images of Western blotting with indicated antibodies to HTT, NeuN, and β-actin. ( e ) Quantification results of mHTT (MW1), total HTT (combining upper and lower bands in the MCA2050 + MCA2051 blot), wtHTT (lower and major band in the blot with MAB 2166 antibody), and NeuN in the striatum injected with AAV-HTT gRNA + Cas9 (HTT gRNA, right striatum-R) or AAV-control gRNA + Cas9 (Con gRNA, left striatum-L). n=3, * p

Techniques Used: CRISPR, Mouse Assay, Sequencing, Fluorescence, Transduction, Western Blot, Injection

9) Product Images from "Pathogen subversion of RIP3-dependent necrosis"

Article Title: Pathogen subversion of RIP3-dependent necrosis

Journal: Cell host & microbe

doi: 10.1016/j.chom.2010.03.006

RIP3 is required, and RIP1 is dispensable for MCMV-associated programmed necrosis (A) Viability of 3T3-SA cells expressing Sc, RIP3-A or RIP3-B shRNAs determined 18 hpi with WT or M45 mut RHIM virus (MOI of 10). (B) Viability of SVEC4-10 cells using a subset of conditions described in (A). (C) IB of 3T3-SA cells infected with WT or M45 mut RHIM virus (MOI of 5), harvested at indicated times for IP of RIP3 followed by detection of vIRA (M45) and RIP3. IB using ~5% of cell lysate to detect vIRA (M45) and β–actin. (D) IB of RIP3+/+, RIP3+/−, and RIP3−/− MEFs to detect RIP3, RIP1, and β–actin. (E) Replication of WT and M45 mut RHIM viruses (MOI of 5) on RIP3+/+ (left panel), RIP3+/− (middle panel), and RIP3−/− (right panel) MEFs over a 72 h time course. Viral titers were determined by plaque assay with the first (0 h) time point representing the amount of virus in the inoculum. (F) IB analysis for FLAG-tagged proteins as well as β-actin (left panel) in RIP3−/− MEFs expressing FLAG-tagged RIP3, RIP3-KD, or RIP3-mRHIM and viability of reconstituted cells (right panel) infected with M45 mut RHIM and WT virus. (G) Viability of RIP3+/+, RIP3+/−, and RIP3−/− MEFs infected with WT or M45 mut RHIM virus in the presence or absence of Nec-1 (30 μM). (H) Viability of RIP3+/+ and RIP3−/− MEFs treated to induce necroptosis as described in Figure 2H in the presence or absence of Nec-1 (30 μM). (I) IB analysis for RIP1 as well as β–actin (top panel) and viability (bottom panel) of WT (RIP3+/+) MEFs stably expressing Sc, RIP1-A or RIP1-B shRNAs. Cell viability was determined for cells infected with WT or either of two independent isolates of M45 mut .

Figure Legend Snippet: RIP3 is required, and RIP1 is dispensable for MCMV-associated programmed necrosis (A) Viability of 3T3-SA cells expressing Sc, RIP3-A or RIP3-B shRNAs determined 18 hpi with WT or M45 mut RHIM virus (MOI of 10). (B) Viability of SVEC4-10 cells using a subset of conditions described in (A). (C) IB of 3T3-SA cells infected with WT or M45 mut RHIM virus (MOI of 5), harvested at indicated times for IP of RIP3 followed by detection of vIRA (M45) and RIP3. IB using ~5% of cell lysate to detect vIRA (M45) and β–actin. (D) IB of RIP3+/+, RIP3+/−, and RIP3−/− MEFs to detect RIP3, RIP1, and β–actin. (E) Replication of WT and M45 mut RHIM viruses (MOI of 5) on RIP3+/+ (left panel), RIP3+/− (middle panel), and RIP3−/− (right panel) MEFs over a 72 h time course. Viral titers were determined by plaque assay with the first (0 h) time point representing the amount of virus in the inoculum. (F) IB analysis for FLAG-tagged proteins as well as β-actin (left panel) in RIP3−/− MEFs expressing FLAG-tagged RIP3, RIP3-KD, or RIP3-mRHIM and viability of reconstituted cells (right panel) infected with M45 mut RHIM and WT virus. (G) Viability of RIP3+/+, RIP3+/−, and RIP3−/− MEFs infected with WT or M45 mut RHIM virus in the presence or absence of Nec-1 (30 μM). (H) Viability of RIP3+/+ and RIP3−/− MEFs treated to induce necroptosis as described in Figure 2H in the presence or absence of Nec-1 (30 μM). (I) IB analysis for RIP1 as well as β–actin (top panel) and viability (bottom panel) of WT (RIP3+/+) MEFs stably expressing Sc, RIP1-A or RIP1-B shRNAs. Cell viability was determined for cells infected with WT or either of two independent isolates of M45 mut .

Techniques Used: Expressing, Infection, Plaque Assay, Stable Transfection

10) Product Images from "A Role for the Receptor for Advanced Glycation End Products in Idiopathic Pulmonary Fibrosis"

Article Title: A Role for the Receptor for Advanced Glycation End Products in Idiopathic Pulmonary Fibrosis

Journal: The American Journal of Pathology

doi: 10.2353/ajpath.2008.070569

$mRAGE is highly expressed in the lung. Membrane fractions from each of the indicated tissues were prepared from untreated wild-type C57BL/6 mice and analyzed by Western blot for mRAGE expression. The PVDF membrane was then stripped and reprobed for β-actin expression as a loading control. Note that mRAGE expression is highest in the lungs.$
Figure Legend Snippet: mRAGE is highly expressed in the lung. Membrane fractions from each of the indicated tissues were prepared from untreated wild-type C57BL/6 mice and analyzed by Western blot for mRAGE expression. The PVDF membrane was then stripped and reprobed for β-actin expression as a loading control. Note that mRAGE expression is highest in the lungs.

Techniques Used: Mouse Assay, Western Blot, Expressing

11) Product Images from "p53-Regulated Increase in Oxidative-Stress-Induced Apoptosis in Fuchs Endothelial Corneal Dystrophy: A Native Tissue Model"

Article Title: p53-Regulated Increase in Oxidative-Stress-Induced Apoptosis in Fuchs Endothelial Corneal Dystrophy: A Native Tissue Model

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.11-8312

Increased p53 protein level in FECD compared with normal CECs. ( A ) A representative Western blot shows increased p53 levels in FECD compared with normal CEC. β-Actin was used for normalization of protein loading. ( B ) Densitometric analysis of

Figure Legend Snippet: Increased p53 protein level in FECD compared with normal CECs. ( A ) A representative Western blot shows increased p53 levels in FECD compared with normal CEC. β-Actin was used for normalization of protein loading. ( B ) Densitometric analysis of

Techniques Used: Western Blot, Capillary Electrochromatography

12) Product Images from "Liver-Specific Ablation of Integrin-Linked Kinase in Mice Results in Enhanced and Prolonged Cell Proliferation and Hepatomegaly after Phenobarbital Administration"

Article Title: Liver-Specific Ablation of Integrin-Linked Kinase in Mice Results in Enhanced and Prolonged Cell Proliferation and Hepatomegaly after Phenobarbital Administration

Journal: Toxicological Sciences

doi: 10.1093/toxsci/kfp281

(A) HGF and its receptor cMet protein expression after PB administration in WT and ILK/liver−/− mice. Pooled liver samples from at least three mice were used for protein. β-Actin was used as the loading control. (B) Semiquantitative

Figure Legend Snippet: (A) HGF and its receptor cMet protein expression after PB administration in WT and ILK/liver−/− mice. Pooled liver samples from at least three mice were used for protein. β-Actin was used as the loading control. (B) Semiquantitative

Techniques Used: Expressing, Mouse Assay

(A) Changes in cell cycle-related proteins in ILK/liver−/− and WT mice after PB administration. Pooled liver samples from at least three mice were used for protein. β-Actin and TATA-binding protein were used as loading controls

Figure Legend Snippet: (A) Changes in cell cycle-related proteins in ILK/liver−/− and WT mice after PB administration. Pooled liver samples from at least three mice were used for protein. β-Actin and TATA-binding protein were used as loading controls

Techniques Used: Mouse Assay, Binding Assay

Immunofluorescence:

Article Title: Ubiquitination-Induced Fluorescence Complementation (UiFC) for Detection of K48 Ubiquitin Chains In Vitro and in Live Cells
Article Snippet: .. The primary antibodies used for immunofluorescence were: rabbit monoclonal anti-K48 ubiquitin chain (clone Apu2) (Millipore), rabbit monoclonal anti-K11 ubiquitin chain (clone 2A3/2E6) (Millipore), rabbit monoclonal anti-K63 ubiquitin chain (clone Apu3) (Millipore) and mouse monoclonal anti-K63 ubiquitin chain antibody (clone HWA4C4) (Millipore), rabbit polyclonal anti-GFP-HRP antibodies (Rockland), mouse monoclonal anti-ubiquitin-HRP antibody (Santa Cruz Biotech), rabbit polyclonal anti-p62/SQSTM1 antibodies (Sigma), mouse monoclonal anti-p62/SQSTM1 antibody and FK2 anti-ubiquitin antibody (Cell Signaling), and rabbit polyclonal anti-Tom20 antibodies (Santa Cruz). .. Secondary antibodies, including anti-mouse and anti-rabbit IgG antibodies conjugated with Alexa Fluor 350 (blue) or Alexa Fluor 594 (red) were purchased from Invitrogen.

Chromatin Immunoprecipitation:

Article Title: Ultraviolet-B induces ERCC6 repression in lens epithelium cells of age-related nuclear cataract through coordinated DNA hypermethylation and histone deacetylation
Article Snippet: .. Chromatin immunoprecipitation (ChIP) Assay ChIP assays were performed by using Magna ChIP TM A/G (Millipore Corporation, Temecula, CA), and the sheared chromatin samples were used for immunoprecipitation with 1 μg of mouse anti-human-Sp1 (Millipore) anti-acety H3K9 (Upstate Biotechnology), rabbit anti-human-HADC1(Millipore), and goat anti-human-DNMT3b (Millipore) antibodies, overnight at 4 °C. .. The eluted DNA and the aliquots of chromatin prior to immunoprecipitation (input) were amplified by RT-PCR.

Immunoprecipitation:

other:

Article Title: Irisin, a Novel Myokine, Regulates Glucose Uptake in Skeletal Muscle Cells via AMPK
Article Snippet: Antibodies against AMPK, phospho-AMPK (Thr172 ), acetyl-CoA carboxylase (ACC), and phosphor-ACC (Ser79 ) were from Millipore-Upstate; p38 MAPK and phospho-p38 MAPK (Thr180 /Tyr182 ) from Santa Cruz; and myogenin was purchased from Epitomics, Inc. Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Enzo Life Sciences.

Western Blot:

Article Title: Sildenafil reduces neuroinflammation and restores spatial learning in rats with hepatic encephalopathy: underlying mechanisms
Article Snippet: For immunofluorescence, sections were incubated with the corresponding fluorescent secondary antibody followed by incubation with the nuclear marker DAPI. .. Primary antibodies used were the same as for Western blot but with (1:200) dilutions for IL-1β, receptor of IL1β, p38-MAPK from Millipore (Darmstadt, Germany). .. The secondary biotinylated antibodies (1:200) from Vector Laboratories (Burlingame, CA, USA) and secondary fluorescent antibodies (1:500) from Invitrogen (Oregon, USA) were used.

Inhibition:

Article Title: Topical p38 MAPK Inhibition Reduces Bacterial Growth on Burn Wounds in an in vivo Burn Model
Article Snippet: .. For topical inhibition of p38 MAPK, 1 mL of 10−4 M of SB202190 (Calbiochem, San Diego, CA) - a specific p38α/β inhibitor- was applied to the wound immediately after injury, and repeated twice every 8h during the first 24h. ..

Modification:

Article Title: Induction of tropomyosin during hepatic stellate cell activation and the progression of liver fibrosis
Article Snippet: Pronase E was obtained from Merck (Damstadt, Germany). .. Mouse monoclonal IgG2a antibody against α-SMA, mouse monoclonal IgG1 antibody against tropomyosin, Dulbecco’s modified Eagle’s medium (DMEM), TAA, and fetal bovine serum (FBS) were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). .. Rabbit polyclonal IgG antibodies against human platelet-derived growth factor receptor-β (PDGFR-β) that reacts with rat PDGFR-β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).