Journal: Viruses

Article Title: Silvestrol Inhibits Chikungunya Virus Replication

doi: 10.3390/v10110592

Figure Lengend Snippet: Analysis of signal transduction during CHIKV infection in the presence of 50 nM silvestrol. ( A ) HEK 293T cells were seeded in six-well plates and were infected with CHIKV using an MOI of 1, and 50 nM silvestrol was added where indicated. Cells were treated with IFNα for 30 min before harvest if indicated, and Western blot analysis of cell lysates was performed. (−) Untreated cells; (+) treated cells. In lanes 3–6, cells were infected with CHIKV. The CHIKV E2 protein, STAT1, eIF2α, and their phosphorylated proteins were detected with specific antibodies and secondary HRP-coupled antibodies, and the ECL detection system (Amersham, Freiburg). Equal loading of each blot was controlled by detection of β-actin; and, ( B ) Uninfected HEK293T cells were treated with IFNα for 30 min before harvest and either treated with silvestrol for 16 h or left untreated. Western blot analysis of cell lysates was performed and p-STAT1 and STAT1 were detected. STAT1 served as a loading control.

Article Snippet: The following conditions were used: 50 mM sodium borate pH 9.0, 20% methanol, and 0.1% SDS buffer at 100 mA per membrane for 75 min. After the blotting, membranes were blocked with Roti-BlockTM (Carl Roth, Karlsruhe, Germany), and specific proteins were detected with antibodies directed against: CHIKV-E2 (Eurogentec, Köln, Germany, custom made), STAT1 (Sigma, Munich, Germany; #HPA000982), phospho-STAT1 (Cell Signaling, Frankfurt am Main, Germany; #7649), eIF2α (R&D Systems, Abingdon, UK; #AF3997), phospho-eIF2α (R&D Systems, Abingdon, UK; #MAB39971), mCherry (Abcam, Cambridge, UK, #ab183628), CHIKV-nsP2 (Abgenex, Bhubaneswar, India; clone ABM3F3.2E10), or β-actin (Sigma, Munich, Germany; #A5441).

Techniques: Transduction, Infection, Western Blot, Control

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Expression of the indicated eIF2α kinase was reduced in LNCaP cells using gene-specific siRNAs. Two different siRNAs were used for knockdown of each eIF2α kinase and compared to scrambled siRNA control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and is plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were transfected with two different siRNAs targeting GCN2 or a scramble siRNA control and cell lysates were prepared and immunoblotted for the indicated proteins. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to scramble siRNA control are indicated. ( C ) Expression of GCN2 was knocked-down in LAPC-4, C4-2B, MR49F, 22Rv1, or PC-3 cells using two different siRNAs and compared to scrambled siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) LNCaP cells were treated with indicated concentrations of GCN2iB and cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤0.0001. ( E ) LNCaP cells were treated with GCN2iB (2 µM) or DMSO control for 24 hr and protein lysates were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin as indicated. Relative levels of p-eIF2α normalized to total eIF2α are shown. ( F ) Levels of p-GCN2 were measured in prostate tumor microarrays (Biomax PR1921b and PR807c) using immunohistochemistry (IHC). Staining for p-GCN2-T899 from non-malignant ( N = 33) and malignant PCa tissue ( N = 88) from patients >50 years old was analyzed and quantified using QuPath to determine the histoscore and is represented as a scatterplot. Statistical significance was determined using an unpaired two-tailed t -test; *p ≤ 0.05. Representative images showing p-GCN2-T899 staining of normal and malignant prostate tissues are shown. Scale bars shown are 200 µm (main image) and 20 µm (insert).

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Expressing, Standard Deviation, Transfection, Molecular Weight, Western Blot, Immunohistochemistry, Two Tailed Test, Staining

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were transfected with the indicated gene-specific siRNAs or a scramble control and the protein levels of HRI, PERK, or PKR and p-eIF2α, total eIF2α, and ATF4 were measured by immunoblot. Measurements of actin were used as a protein loading control in the immunoblot experiments. Molecular weight markers are shown in kilodaltons. ( B ) The indicated PCa cell lines were transfected with siRNAs targeting GCN2 or ATF4. Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, or actin as indicated. ATF4 was not detected in the LAPC-4 cells.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Transfection, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Immunoblot analyses for 22Rv1, PC-3, and GCN2 KO clones. Protein lysates were analyzed by immunoblot to measure the levels of GCN2, ATF4, p-eIF2α, total eIF2α, or actin. Molecular weight markers are shown in kilodaltons. The relative levels of p-eIF2α normalized to total eIF2α compared to wild-type (WT) parental control are indicated. ( B ) Growth curve of 22Rv1 WT, 22Rv1 GCN2 KO (clone 7), or 22Rv1 GCN2 KO (clone 7) cells overexpressing GCN2. Data from replicate wells ( N = 5) are shown as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( C ) Protein lysates prepared from the 22Rv1 WT, 22Rv1 GCN2 KO (clone 7), or 22Rv1 GCN2 KO (clone 7) cells expressing GCN2 were analyzed by immunoblot for GCN2, ATF4, ASNS, or actin.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Clone Assay, Molecular Weight, Standard Deviation, Expressing

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) C4-2B or 22Rv1 cells, cultured as indicated in the Materials and methods, or PC-3 cells cultured in HPLM media were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( B ) 22Rv1 WT and 22Rv1 GCN2 KO (clone 7) cells were treated with GCN2iB as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± SD) relative to day 0. Statistical significance was determined using a two-way ANOVA as described in ; ****p ≤ 0.0001. ( C ) Lysates were prepared from C4-2B, 22Rv1, or PC-3 cells treated with GCN2iB at the indicated concentrations or vehicle control (dimethyl sulfoxide, DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2) AR, or actin. Molecular weight markers are indicated in kilodaltons.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Cell Culture, Standard Deviation, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Lysates were prepared from BPH-1, LNCaP C4-2B, 22Rv1, or PC-3 cells and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, AR, or actin. Molecular weight markers are indicated in kilodaltons. ( B ) BPH-1 cells were transfected with siRNAs targeting GCN2, ATF4, or 4F2 (SLC3A2). Protein lysates were prepared and analyzed by immunoblot to determine the levels of GCN2, ATF4, 4F2 (SLC3A2), or actin as indicated. Molecular weight markers are indicated in kilodaltons. ( C ) Expression of GCN2, ATF4, or 4F2 (SLC3A2) was reduced in BPH-1 cells using two different gene-specific siRNAs as indicated and compared to a scramble siRNA control. Cell growth was measured for up to 6 days in replicate wells ( N = 5) as described in A . Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; *p ≤ 0.05, **p ≤ 0.01. ( D ) Lysates were prepared from BPH-1 cells treated with GCN2iB at the indicated concentrations or vehicle control (DMSO) for 48 hr and immunoblot analysis was carried out using antibodies that recognize p-GCN2-T899, total GCN2, p-eIF2α-S51, total eIF2α, ATF4, ASNS, TRIB3, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), AR, or actin. Molecular weight markers are indicated in kilodaltons. ( E ) BPH-1 cells were treated with 0.5–10 µM GCN2iB or vehicle (DMSO) control as indicated for up to 6 days. Cell growth was measured ( N = 5) and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way ANOVA is shown in .

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Molecular Weight, Transfection, Expressing, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Amino acid measurements of LNCaP cells treated with 2 µM GCN2iB or vehicle control (DMSO) for 8 hr. Bar graphs in the top panel show high abundance amino acids and the lower panel those with lower levels. The heat map on the right shows fold change in amino acid abundance for each biological replicate of GCN2iB-treated LNCaP cells versus the vehicle with the scale showing the highest fold change in yellow and lowest in purple. Statistical significance was determined using an unpaired two-tailed t -test. Error bars indicate standard deviation (SD) ( N = 3); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. ( B ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), vehicle + essential amino acids (EAA), or GCN2iB (2 µM) + EAA, and cell growth was measured for up to 6 days. Error bars indicate SD ( N = 5). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( C ) Cell cycle analyses of LNCaP cells treated with vehicle, GCN2iB (2 µM), vehicle + EAA, or GCN2iB (2 µM) + EAA for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( D ) Genome-wide tRNA charging analysis (CHARGE-seq) of LNCaP cells treated with vehicle (DMSO), GCN2iB (2 µM), or GCN2iB (2 µM) + EAA for 8 hr. The tRNA charging ratio is shown as a bar graph with fold change compared to vehicle. Only tRNA isoacceptors measured in LNCaP cells are shown. Error bars indicate SD ( N = 4). ( E ) tRNA charging percentage for tRNA His in LNCaP cells treated with vehicle, GCN2iB, or GCN2iB + EAA. Statistical significance was determine using a one-way ANOVA with Tukey’s multiple comparisons ( N = 4); ***p ≤ 0.001, ****p ≤ 0.0001. ( F ) LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + EAA, or GCN2iB (2 µM) combined with the indicated individual amino acids. Cell growth was measured at 4 days in triplicate wells ( N = 3). Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD; ****p ≤ 0.0001. ( G ) Cell cycle analysis of LNCaP cells were treated with vehicle, GCN2iB (2 µM), GCN2iB (2 µM) + histidine (200 µM), or with media lacking histidine for 48 hr. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons. Error bars indicate SD ( N = 3); ***p ≤ 0.001, ****p ≤ 0.0001. ( H ) LNCaP cells were cultured in normal media, media supplemented with EAA mix, or media supplemented with histidine (200 µM) for 24 hr. Lysates were analyzed by Immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, or actin. Molecular weight markers are presented in kilodaltons for each immunoblot panel. The relative levels of p-eIF2α normalized to total eIF2α compared to normal media (NM) control are indicated.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Two Tailed Test, Standard Deviation, Genome Wide, Cell Cycle Assay, Cell Culture, Western Blot, Molecular Weight

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Gene-level depletion for LNCaP and 22Rv1 cells. The average log2 fold change for the single guide RNAs (sgRNAs) for each gene is shown on the x -axis. Significantly depleted genes (p ≤ 0.05) in LNCaP or 22Rv1 are indicated. Circle size indicates the number of significant sgRNAs. SLC genes in red are dependent on GCN2 for expression. ( B ) Plot of −Log 10 (p value) for depleted genes identified in CRISPR screen for LNCaP versus 22Rv1 cells. Significantly depleted genes (p ≤ 0.05) in LNCaP, 22Rv1 or both cell lines are indicated. SLC genes in red are GCN2 dependent. ( C ) Lysates from LNCaP cells were treated with 2 µM GCN2iB for 6 or 24 hr, or with vehicle (DMSO) were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. Molecular weight markers are indicated in kilodaltons for the panels. ( D ) LNCaP cells were cultured in standard culture conditions (NM: normal media), media supplemented with 200 µM histidine (+His), or media depleted of histidine (−His) for 24 hr. Lysates were analyzed by immunoblot analyses using antibodies that recognize total or phosphorylated GCN2-T899, ATF4, 4F2 (SLC3A2), or actin. ( E ) LNCaP cells were treated with 100 nM halofuginone (HF) for 2 and 6 hr or vehicle (DMSO). Lysates were analyzed by Immunoblot using antibodies that recognize the indicated proteins. ( F ) 4F2 (SLC3A2) expression was reduced in LNCaP or 22Rv1 cells using two different siRNAs or scramble siRNA as a control. Cell growth was measured in replicate wells ( N = 5) for up to 6 days and are plotted relative to day 0 (mean ± standard deviation [SD]). Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001. ( G ) LNCaP cells transfected with two different siRNAs targeting 4F2 (SLC3A2) or scramble siRNA for 48 hr. Lysate was prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2-T899, total or phosphorylated eIF2α−S51, ATF4, 4F2 (SLC3A2), or actin. ( H ) LNCaP cells stably overexpressing SLC3CA2 or vector control were transfected with two different siRNAs targeting GCN2 or scrambled control. Cells were then treated with GCN2iB (2 µM) or vehicle and growth was measured in replicate wells ( N = 5) and is plotted relative to day 0 (mean ± SD). Statistical significance was determined using a two-way ANOVA as described in ; **p ≤ 0.01, ****p ≤ 0.0001. ( I ) Amino acid measurements of LNCaP cells transfected siRNA targeting GCN2 ( N = 4), 4F2 (SLC3A2, N = 4), or scramble control ( N = 8). Two separate bar graphs show high abundance (top) and low abundance (bottom) amino acids. Statistical significance was determined using a two-way ANOVA as described in . Error bars indicate SD; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Expressing, CRISPR, Western Blot, Molecular Weight, Cell Culture, Standard Deviation, Transfection, Stable Transfection, Plasmid Preparation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) LNCaP cells were treated with GCN2iB (2 µM) or vehicle (DMSO) control in the presence or absence of salubrinal (50 µM) for 48 hr. Protein lysates were prepared and analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, 4F2 (SLC3A2), or actin as indicated. ( B ) LNCaP cells transfected with empty vector (EV) control or pMSCV-GADD34-puro expression plasmid encoding the human GADD34 gene were analyzed by immunoblot as indicated in panel A. ( C ) Protein lysates prepared from LNCaP or 22Rv1 stably expressing empty vector (EV) control or 4F2 (SLC3A2) were analyzed by immunoblot using antibodies that recognize total or phosphorylated GCN2, total or phosphorylated eIF2α(S-51), ATF4, or actin as indicated. ( D ) Growth of LNCaP and 22Rv1 cells stably expressing empty vector (EV) control or 4F2 (SLC3A2) was measured in replicate wells ( N = 5) for up to 4 days and plotted as fold change (mean ± standard deviation [SD]) relative to day 0. Statistical significance was determined using a two-way analysis of variance (ANOVA) as described in ; ****p ≤ 0.0001.

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Transfection, Plasmid Preparation, Expressing, Stable Transfection, Standard Deviation

Journal: eLife

Article Title: GCN2 eIF2 kinase promotes prostate cancer by maintaining amino acid homeostasis

doi: 10.7554/eLife.81083

Figure Lengend Snippet: ( A ) Lysates from 22Rv1 WT, 22Rv1 GCN2 KO, and 22Rv1 ATF4 KO tumors were subjected to immunoblot analyses to measure total or phosphorylated GCN2, total or phosphorylated eIF2α, ATF4, LAT1 (SLC7A5), xCT (SLC7A11), 4F2 (SLC3A2), CAT1 (SLC7A1), ASCT1 (SLC1A4), ASCT2 (SLC1A5), androgen receptor (AR), AR splice variant 7 (AR-V7), or actin. Molecular weight markers are indicated in kilodaltons for each immunoblot panel. Levels of the indicated proteins normalized to appropriate control are shown in the bar graph on the right. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons. Error bars indicate standard deviation (SD) ( N = 4); ns, p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001. ( B ) LNCaP cells were transfected with siRNAs targeting GCN2 ( N = 4), ATF4 ( N = 4), or scramble control ( N = 8) for 48 hr. Amino acid levels were determined as described in the Materials and methods. Error bars indicate SD. Statistical significance was determined using a one-way ANOVA with Tukey’s multiple comparisons; # p ≤ 0.1, *p ≤ 0.05, **p ≤ 0.01, ***p ≤0.001, ****p ≤0.0001. Scramble control and GCN2 knockdown samples are the same as in .

Article Snippet: The primary antibodies used were as follows: phospho-GCN2-T899 (Abcam Cat. #ab75836, RRID: AB_1310260 ), total GCN2 (Cell Signaling Technology Cat. #3302, RRID: AB_2277617 ), total PERK (Cell Signaling Technology Cat. #3192, RRID: AB_2095847 ), total HRI (Santa Cruz Biotechnology Cat. #sc-365239, RRID: AB_10843794 ), total PKR (Cell Signaling Technology Cat. #12297, RRID: AB_2665515 ), phospho-eIF2α-S51 (Abcam Cat. #ab32157, RRID: AB_732117 ), total eIF2α (Cell Signaling Technology Cat. #5324, RRID: AB_10692650 ), ATF4 (Cell Signaling Technology Cat. #11815, RRID: AB_2616025 ), or custom rabbit polyclonal antibody which was prepared against full-length recombinant human ATF4 protein and affinity purified, ASNS (Cell Signaling Technology, Cat. #20843S), TRIB3 (Abcam Cat. #ab75846, RRID: AB_1310768 ), GADD34 (Proteintech Cat. #10449-1-AP, RRID: AB_2168724 ), SLC7A5/LAT1 (Cell Signaling Technology Cat. #5347, RRID: AB_10695104 ), SLC7A11/xCT (Cell Signaling Technology Cat. #12691, RRID: AB_2687474 ), SLC3A2/4F2 (Cell Signaling Technology Cat. #47213, RRID: AB_2799323 ), SCL7A1/CAT1 (Proteintech Cat. #14195-1-AP, RRID: AB_2190723 ), SLC1A4/ASCT1 (Cell Signaling Technology Cat. #8442, RRID: AB_10828382 ), SLC1A5/ASCT2 (Cell Signaling Technology Cat. #5345, RRID: AB_10621427 ), AR (Cell Signaling Technology Cat. #5153, RRID: AB_10691711 ), puromycin (Millipore Cat. #MABE343, RRID: AB_2566826 ), β-actin (Sigma-Aldrich Cat. #A5441, RRID: AB_476744 ), and β-tubulin (Cell Signaling Technology Cat. #2146, RRID: AB_2210545 ).

Techniques: Western Blot, Variant Assay, Molecular Weight, Standard Deviation, Transfection

Journal: Methods (San Diego, Calif.)

Article Title: Methods for the characterization of stress granules in virus infected cells

doi: 10.1016/j.ymeth.2015.04.009

Figure Lengend Snippet: Commonly used antibodies for SG components.

Article Snippet: eIF2α total , Cell Signaling 2103S , Mouse , , Present in Pat A SGs, not arsenite SGs.

Techniques: Labeling, Marker

Journal: Nature Neuroscience

Article Title: Neuronal activity rapidly reprograms dendritic translation via eIF4G2:uORF binding

doi: 10.1038/s41593-024-01615-5

Figure Lengend Snippet: a , Immunofluorescence (IF) images of primary cortical neurons immunostained for glial fibrillary acidic protein (GFAP) and oligodendrocyte transcription factor 2 (OLIG2) simultaneously with PSD95 to show that the cultures are devoid of glial cells or oligodendrocytes, respectively. DAPI for nuclei; PSD95 for excitatory neurons. Magnification, ×40. Scale bars, 50 μm. b , TurboID-PSD95 was cloned without (top row) and with (bottom row) its 5′ and 3′ UTRs and lentivirally expressed in primary cortical neurons. White dashed boxes are zoomed in areas in black&white images. DAPI for nuclei; MAP2 for dendrites; Flag for each TurboID. % dendritically localized TurboID-PSD95 is quantified by co-localization with MAP2 signal in ImageJ. 3 different areas of images per replicate ( n = 3). Magnification, ×20. Scale bars, 50 μm. Significance was derived from biological replicates, showing the center line at mean. c , IF images of TurboID-PSD95-transduced neurons immunostained for DAPI (blue, for nuclei), PSD95 (red, for endogenous PSD95) and TurboID-PSD95 (cyan, detected by Flag). Magnification, ×60. Scale bar, 50 μm. d , IF images show the expression of a presynaptic marker, Synaptophysin (cyan), and TurboID-PSD95 (red, detected by Flag antibody) in primary cortical neurons transduced with TurboID-PSD95. DAPI (blue) marker for nuclei. Three zoomed in regions are marked by the white boxes. Magnification, ×60. Scale bar, 10 μm. e , IF images show TurboID expression and biotinylation in primary cortical neurons transduced with TurboID-PSD95 or Pan-TurboID after 30 minutes of biotin incubation. DAPI (blue, nuclei); MAP2 (green, dendrites); Flag (red, TurboID); and Streptavidin (cyan, biotinylated proteins). Magnification, ×20. Scale bars, 50 μm. f , Western blots stained for Flag and β-Actin from Pan-TurboID and TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin shown to indicate the relative expression levels of TurboID proteins. Quantifications of TurboID protein levels normalized to β-Actin are shown on the right ( n = 3); relative levels are not significant by two-tailed, paired Student’s t -test. g , Western blots stained for streptavidin signal in inputs (‘in’) and streptavidin pulldowns (‘pd’) from Pan-TurboID or TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. h , Streptavidin pulldowns shown for dendritic (SHANK3, GKAP, NLGN1 and HOMER1) and negative control (GAPDH) proteins from TurboID-PSD95-transduced neurons in the absence (−) or presence (+) of exogenous biotin. Flag signal indicates self-biotinylation of each construct. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by TurboID-PSD95 in each condition is calculated by dividing the signal in the pulldown lane by that of the input lane, after each is adjusted to total, and quantifications are shown as bar graphs ( n = 3). P values: Flag = 0.58, SHANK3 = 0.0061, GKAP = 0.018, NLGN1 = 0.00052, HOMER1 = 0.021, GAPDH = 0.42. i , Streptavidin pulldowns shown for dendritic (BAIAP2 and DLGAP3) and nuclear (TBR1, H4 and H2AX) proteins from Pan-TurboID and TurboID-PSD95-transduced neurons in the presence (+) of exogenous biotin. Loaded on the gel are 10% (by volume) of input and 50% (by volume) of pulldowns. Percent isolated by each TurboID is calculated as in (h) ( n = 3). P values: BAIAP2 = 0.0052, DLGAP3 = 0.0035, TBR1 = 0.0063, H4 = 0.018, H2AX = 0.0037. j , Phosphorylation of EEF2, eIF2α, ERK1/2 and IRE1 and total levels of ATF4 and CHOP are shown in resting (rest), activated (DHPG, Dep) and stressed (Sodium arsenite (NaAsO 2 )) cells by using phospho-specific and total antibodies. The amount of phosphorylated or total protein is shown in the bar graphs, calculated by dividing the phosphorylated signal to total and β-Actin for the phosphorylated proteins and by dividing the total to β-Actin for ATF4 and CHOP ( n = 3). Significance was calculated with respect to rest. P values: P-EEF2 (DHPG = 0.0088, Dep = 0.0023, NaAsO 2 = 0.039), P-eIF2α (DHPG = 0.018, Dep = 0.0034, NaAsO 2 = 0.028), P-ERK1/2 (DHPG = 0.015, Dep = 0.0067, NaAsO 2 = 0.00084), P-IRE1 (DHPG = 0.06, Dep = 0.37, NaAsO 2 = 0.0027), ATF4 (DHPG = 0.038, Dep = 0.42, NaAsO 2 = 0.016), CHOP (DHPG = 0.044, Dep = 0.18, NaAsO 2 = 0.024). k , Quantitative PCR (qPCR) results shown for immediate early genes, Arc , Fos and Jun . The fold changes for each gene are calculated by first normalizing to the house-keeping gene β-Actin in each condition, then dividing the value of each condition by that of the resting state ( n = 3). l , Dendritic spine size in resting and KCl-depolarized neurons are measured using the Keyence microscope. Red squares are examples of spines that are counted ( n = 3, 12 spines from each biological replicate are counted as technical replicates). Significance was derived from the biological replicates using the two-tailed, unpaired Student’s t -test. Box plots show the min and max, with the center line at median. Magnification, ×100. Scale bars, 5 μm. m , Fluo-4-AM staining in resting, KCl-depolarized and DHPG-depolarized cells. Fluo4-AM was loaded in resting cells and measurements were taken at indicated time points after Fluo4-AM removal. In depolarized cells, the dye was loaded during silencing. After silencing, fluorescence was measured during stimulus at 10, 30 and 60-minute time points for the KCl treatment and at 10-minute for the DHPG-induced activation. Fluorescence was also measured 60 minutes after the stimulus removal (60′post KCl and 60′post DHPG). Circles represent data from 2 biological and 3 technical replicates. Below: Examples of Fluo4-AM fluorescence are shown in resting, 10-minute KCl-treated and 10-minute DHPG-treated neurons. Fluo4-AM loading (45 minutes) was performed during the last 45 minutes of the silencing step prior to stimulus addition for the KCl and DHPG treatment and simultaneously for the resting neurons. Imaging was performed 10 minutes after the stimulus was added. Scale bars, 50 μm. (b,f,h-k,m) Data are mean ± s.d. Significance was calculated using the two-tailed, paired Student’s t -test. P values: ns (not significant) >0.05; * <0.05; ** <0.01; *** <0.001; **** <0.0001. n indicates the number of biologically independent samples.

Article Snippet: Puromycin (1:3,000, mouse, Kerafast, EQ0001, RRID: AB_2620162), Flag (1:3,000, mouse, Sigma-Aldrich, F1804, RRID: AB_262044), β-Actin antibody (1:2,500, mouse, Sigma-Aldrich, A1978, RRID: AB_476692), RPL10A (1:1,000, rabbit, Abcam, ab174318), MAP2 (1:2,500, guinea pig, Synaptic Systems, 188004, RRID: AB_2138181), GFAP (1:500, rabbit, Abcam, ab7260, RRID: AB_305808), OLIG2 (1:500, rabbit, Proteintech, 13999-1-AP, RRID: AB_2157541), PSD95 (1:500, mouse, Millipore, MABN68, RRID: AB_10807979), Synaptophysin (1:300, mouse, Abcam, ab8049, RRID: AB_2198854), SHANK3 (1:500, mouse, Novus, NBP1-47610, RRID: AB_10010567), GKAP (1:500, rabbit, Novus, NBP1-76911, RRID: AB_11017331), NLGN1 (1:200, mouse, Novus, NBP2-42192), HOMER1 (1:1,000, rabbit, Proteintech, 12433-1-AP, RRID: AB_2295573), GAPDH (1:5,000, mouse, Thermo Fisher Scientific, AM4300, RRID: AB_2536381), BAIAP2 (1:500, rabbit, Proteintech, 11087-2-AP, RRID: AB_2063075), DLGAP3 (1:500, rabbit, Proteintech, 55056-1-AP, RRID: AB_10858793), TBR1 (1:500, rabbit, Proteintech, 20932-1-AP, RRID: AB_10695502), H4 (1:1,000, mouse, Abcam, ab31830, RRID: AB_1209246), H2A.X (1:1,000, rabbit, Proteintech, 10856-1-AP, RRID: AB_2114985), EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2332, RRID:AB_10693546), P-EEF2 (1:1,000, rabbit, Cell Signaling Technology, 2331, RRID: AB_10015204), eIF2α (1:1,000, rabbit, Cell Signaling Technology, 9722, RRID: AB_2230924), P-eIF2α (1:1,000, rabbit, Cell Signaling Technology, 3398, RRID: AB_2096481), p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 4695, RRID: AB_390779), P-p42/44 MAPK (1:1,000, rabbit, Cell Signaling Technology, 9101, RRID: AB_331646), P-IRE1 (1:500, rabbit, Novus, NB100-2323SS, RRID: AB_10145203), IRE1 (1:500, rabbit, Novus, NB100-2324SS, RRID: AB_10000972), CHOP (1:1,000, mouse, Cell Signaling, 2895T, RRID: AB_2089254), ATF4 (1:1,000, rabbit, Cell Signaling Technology, 11815S, RRID: AB_2616025), MPHOSPH (1:300, rabbit, Biorbyt, orb100446), KCNJ9 (1:300, rabbit, LSBio, LS-C352416), KCNJ9 (1:300, mouse, Antibodies Incorporated, 75-445, RRID: AB_2686912), eIF4G2 (1:1,000, rabbit, Cell Signaling Technology, RRID: AB_10622189 and rabbit, Cell Signaling Technology, RRID: AB_2261993), NSUN3 (1:250, rabbit, LSBio, LS-C163024), MTF1 (1:300, rabbit, Novus, NBP1-86380, RRID: AB_11011361), ZFP64 (1:300, rabbit, Proteintech, 17187-1-AP, RRID: AB_2218826) and KATNBL1 (1:250, rabbit, Proteintech, 24795-1-AP, RRID: AB_2879730).

Techniques: Immunofluorescence, Clone Assay, Derivative Assay, Expressing, Marker, Transduction, Incubation, Western Blot, Staining, Two Tailed Test, Negative Control, Construct, Isolation, Real-time Polymerase Chain Reaction, Microscopy, Fluorescence, Activation Assay, Imaging

Journal: Circulation Research

Article Title: Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

doi: 10.1161/circresaha.107.154781

Figure Lengend Snippet: Figure 2. Effects of RuCO on PERK, eIF2, ATF4, HO-1, and Nrf2 in HUVECs. A, Cells were exposed to 20 mol/L RuCO, 10 mol/L TG, or 10 mol/L TM for indicated periods of time. B, Cells were exposed to 20 mol/L RuCO or 20 mol/L arsenite (positive control) for 160 minutes. C, Cells transfected with siRNA against eIF2 were exposed to RuCO for 1 hour. D, Cells were exposed for 1 hour (PERK and eIF2 phosphorylations) or 6 hours (ATF4 expression) to RuCO in the presence or absence of 50 g/mL Hb. E, Cells trans- fected with ether PERK siRNA or eIF2 siRNA were exposed to RuCO for 1 hour (Nrf2 nuclear translocation) or 6 hours (HO-1 expres- sion). NF indicates nuclear fraction. Western blot analysis and densitometry analysis were performed as described in Materials and Methods. Blots shown are representative of 3 independent experiments. Values are meansSD from 3 independent experiments. *P0.05 with respect to each untreated group.

Article Snippet: Predesigned siRNAs against human HO-1 (catalog no. SC-35554), PERK (catalog no. SC-36213), eIF2 (catalog no. SC-35272), CHOP (catalog no. SC-35437), and control scrambled siRNA (catalog no. SC-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Positive Control, Transfection, Expressing, Translocation Assay, Western Blot

Journal: Circulation Research

Article Title: Carbon Monoxide Induces Heme Oxygenase-1 via Activation of Protein Kinase R–Like Endoplasmic Reticulum Kinase and Inhibits Endothelial Cell Apoptosis Triggered by Endoplasmic Reticulum Stress

doi: 10.1161/circresaha.107.154781

Figure Lengend Snippet: Figure 6. Effects of RuCO and RuCO-induced HO-1 on TG-, TM-, and HCys-induced CHOP in HUVECs. A, Cells were preincubated for 6 hours with 20 mol/L RuCO and then were exposed for 6 hours to 10 mol/L TG, 10 mol/L TM, or 50 mol/L HCys. B, Cells trans- fected with either PERK siRNA or eIF2 were preincubated for 6 hours without or with RuCO and then were exposed to TG for 6 hours. C, Normal cells and the cells transfected with siRNA against HO-1 were preincubated for 6 hours with RuCO or 10 mol/L CoPP in the presence or absence of 50 g/mL Hb and then exposed to TG for 6 hours. D, Normal cells and the cells transfected with PERK siRNA, HO-1 siRNA, or eIF2 siRNA were preincubated for 6 hours with or without RuCO and then were exposed to TG for 18 hours. Western blot analysis for CHOP, HO-1, ATF6, and Xbp-1 and MTT assay for cell viability were performed as described in Materi- als and Methods. Blots shown are representative of 3 independent experiments. Each bar represents meanSD from 3 independent experiments. *P0.05 with respect to each untreated group. *P0.05.

Article Snippet: Predesigned siRNAs against human HO-1 (catalog no. SC-35554), PERK (catalog no. SC-36213), eIF2 (catalog no. SC-35272), CHOP (catalog no. SC-35437), and control scrambled siRNA (catalog no. SC-37007) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Western Blot, MTT Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: EV-A71 infection results in the phosphorylation and cleavage of PKR. (A) Time course of cellular protein phosphorylation and protein levels after EV-A71 infection. RD cells were infected with EV-A71 at an m.o.i. of 10. At the indicated times post-infection, cell extracts were collected. The total cellular protein in the extracts was quantified, and equivalent amounts from each sample were subjected to western blot analysis for the detection of cellular PKR, phosphorylated-PKR (PKR-p), eIF2α, and phosphorylated eIF2α (eIF2α-p), as well as the expression of viral 3CD and 3C proteins. eIF2α expression was detected as a protein loading control. An asterisk marks non-specific bands. A representative result from three independent experiments is shown. (B) Cleavage of PKR after EV-A71 infection. RD cells were infected with EV-A71 at an m.o.i. of 10 or treated with STS for 2, 4, 6, 8, and 10 h. Cell extracts were collected at the indicated times for immunoblotting analysis to detect the cellular PKR and viral 3A protein levels. PARP cleavage was examined as an apoptosis marker. GAPDH expression was detected as a protein loading control. Asterisks indicate non-specific bands. A representative result from at least three reproducible experiments is shown.

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Infection, Phospho-proteomics, Western Blot, Expressing, Control, Marker

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: EV-A71 3C associates with PKR and induces PKR phosphorylation. (A) The 293T cells were cotransfected with plasmids encoding GFP-PKR-K296H and 3C-Flag, or 3C mutant C147S-Flag, and then harvested at 24 h post-transfection. The cell lysates were immunoprecipitated with antibody against Flag. Samples were then subjected to western blot analysis with detection using anti-GFP and anti-Flag antibodies. * Denotes the heavy chains. (B) Mutations of the protease catalytic sites H40 and C147 of 3C caused loss of the PKR phosphorylation activity. RD cells were either transfected with 3C-Flag, H40D, C147S, H40D/C147S, or R84Q, for 24 h or infected with EV-A71/2231 at an m.o.i. of 10 for 8 h. Cellular extracts were collected and immunoblotting was performed for detecting PKR, PKR-p, eIF2α, eIF2α-p, CstF64, Flag, and viral 3C expression levels. CstF64 cleavage was used as a control for 3C catalytic activity. A representative result from three independent experiments is shown. (C) The PKR inhibitor 2-AP has no effect on 3C-induced apoptosis. The 3C- or H40D/C147S-transfected 293T cells were incubated with or without 2-AP for 24 h and then analyzed for apoptosis by flow cytometry using Annexin V and PI staining. The values shown in the lower left, lower right, and upper right quadrants of each panel represent the percentage of viable, apoptotic, and dead cells, respectively. Data are the means ± SD of values from three independent experiments.

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Phospho-proteomics, Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Activity Assay, Infection, Expressing, Control, Incubation, Flow Cytometry, Staining

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: Expression of a PKR kinase-dead mutant results in an increase of viral proteins and virus titer. (A) RD cells were transiently expressed with PKR or the K296H mutant for 24 h, and then infected with EV-A71 at an m.o.i. of 10. Immunoblot analysis was performed for detecting the presence and phosphorylation of PKR (anti-PKR, anti-PKR-p) and eIF2α (anti-eIF2α, anti-eIF2α-p), and the expression of viral 3A and 3C proteins. GAPDH expression was used as a protein loading control. A representative result from three independent experiments is shown. (B) Stable RD cells expressing vector alone, PKR, or K296H were selected by addition of 3 μg/mL puromycin. Cells were infected with EV-A71 at an m.o.i. of 10 and then the cellular extracts were harvested at 0, 6, and 8 h post-infection. Immunoblot analysis was performed to detect the presence of PKR and viral 3A and 3C proteins. A representative result based on three independent experiments is shown. (C) RD cells stably expressing PKR or the K296H mutant were infected with EV-A71 at an m.o.i. of 10 of for 8 h. The RD cells and culture supernatant were harvested for virus titer determination by plaque assay. The results are expressed as the mean ± SD ( n = 3). * p < 0.05.

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Expressing, Mutagenesis, Virus, Infection, Western Blot, Phospho-proteomics, Control, Plasmid Preparation, Stable Transfection, Plaque Assay

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: dsRNA Binding Domain of PKR Is Proteolytically Released by Enterovirus A71 to Facilitate Viral Replication

doi: 10.3389/fcimb.2017.00284

Figure Lengend Snippet: Proposed model of modulation of PKR function by EV-A71 3C protease. PKR consists of two dsRNA-binding motifs (dsRBM1 + dsRBM2 in green) and the C-terminal kinase domain (gray). In general, binding of viral dsRNA leads to dimerization and autophosphorylation of PKR. Active PKR subsequently phosphorylates its substrate eIF2α, which results in translation inhibition and apoptosis (left). Overexpression of the PKR-K296H mutant (cartoon molecule with a red x) competes with endogenous PKR for dsRNA binding to attenuate PKR activation (middle). In EV-A71 infection, 3C interacts with PKR, which may block its dimerization. Then, 3C cleaves PKR to release dsRNA-binding motifs, which may compete with PKR for the recognition of dsRNA, thereby attenuating PKR activation and increasing viral replication (right).

Article Snippet: Mouse monoclonal antibody against green fluorescent protein (GFP) (SC-9996) and rabbit polyclonal antibodies against eIF2α (SC-11386) and poly (ADP-ribose) polymerase (PARP) (SC-7150) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Binding Assay, Inhibition, Over Expression, Mutagenesis, Activation Assay, Infection, Blocking Assay

Journal: Experimental hematology

Article Title: Panobinostat and venetoclax enhance the cytotoxicity of gemcitabine, busulfan, and melphalan in multiple myeloma cells

doi: 10.1016/j.exphem.2020.01.003

Figure Lengend Snippet: List of primary antibodies, their sources and dilutions

Article Snippet: eIF2α , Cell Signaling/9722 , Rabbit , 2500.

Techniques:

Journal: BioMed Research International

Article Title: Baicalein Induces Apoptosis and Autophagy via Endoplasmic Reticulum Stress in Hepatocellular Carcinoma Cells

doi: 10.1155/2014/732516

Figure Lengend Snippet: Diverse roles of UPR proteins in baicalein-induced apoptosis.(a) SMMC-7721 cells were transfected with scrambled RNA (NC) or CHOP-targeting siRNA (si-CHOP) for 48 h and treated with 0, 100, and 200 μ M baicalein for 24 h. Protein levels of cleaved PARP and CHOP were determined by western blotting. (b) SMMC-7721 cells were transfected with scrambled RNA (NC) or eIF2 α -targeting siRNA (si-eIF2 α ) and then treated with 0, 100, and 200 μ M baicalein for 24 h. Protein levels of cleaved PARP phosphorylated eIF2 α and eIF2 α were determined. (c) After being transfected with scrambled RNA (NC) or IRE1 α -targeting siRNA (si-IRE1 α ), SMMC-7721 cells were treated with the indicated dose of baicalein for 24 h and subjected to western blotting to analyze the level of cleaved PARP, IRE1 α , phosphorylated JNK, and total JNK. GAPDH served as a loading control.

Article Snippet: siRNAs against human eIF2 α , CHOP, IRE1 α , Beclin 1, and Atg5 were synthesized by GenePharma (Shanghai, China).

Techniques: Transfection, Western Blot, Control