Journal: The Journal of Biological Chemistry

Article Title: Secreted Hsp90 Is a Novel Regulator of the Epithelial to Mesenchymal Transition (EMT) in Prostate Cancer *

doi: 10.1074/jbc.M112.389015

Figure Lengend Snippet: Modest elevation of eHsp90 is sufficient to suppress E-cadherin function and promote cell motility. A, upper panel, ELISA analysis of secreted eHsp90 protein detected from conditioned media collected from parental ARCaPE cells stably transduced with control (lacZ) or V5-tagged eHsp90α lentivirus. Lower panel, immunoblot detection of total (endogenous and exogenous) eHsp90α, or V5 detection of transduced eHsp90 protein. B, immunoblot analysis of cell lysates from ARCaPE-LacZ or ARCaPE-eHsp90 confirmed consistent levels of intracellular eHsp90α (IC Hsp90). Indicated analysis of E- and N-cadherin and ERK activity. C, representative morphology of the indicated ARCaPE cells. Analysis of cell motility of ARCaPE-eHsp90 either untreated or treated with NPGA. D, effect of NPGA upon E-cadherin expression in ARCaPE-eHsp90. E, analysis of E-cadherin localization in ARCaPE-LacZ and ARCaPE-eHsp90 untreated cells, or treated for the indicated times with NPGA. F, membrane localization of ZO1 in ARCaPE-LacZ and ARCaPE-eHsp90 untreated cells, or treated with NPGA for 3 days. Asterisks (*) indicate significance of p value ≤0.05. Scale bar is 50 μm.

Article Snippet: V5 antibody (NB600-381) was from Novus Biologicals.

Techniques: Enzyme-linked Immunosorbent Assay, Stable Transfection, Transduction, Western Blot, Activity Assay, 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 ) 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: Journal of Biological Chemistry

Article Title: Nrf1-mediated transcriptional regulation of the proteasome requires a functional TIP60 complex

doi: 10.1074/jbc.ra118.006290

Figure Lengend Snippet: Figure 4. Nrf1 interacts with the TIP60 complex. (A) Wild-type (WT) and Nrf1-/- NIH-3T3 cell lines were treated with 200 nM carfilzomib (CFZ) for 8 hours. The cells were then subjected to chromatin immunoprecipitation (ChIP) with one of IgG, Nrf1, RUVBL1, or TIP60 antibodies. These samples were then analyzed by quantitative PCR with primers specific for antioxidant response element (ARE)- containing promoter regions of proteasome genes PSMA7, PSMB7, and PSMD12. Error bars denote SD (n=3). (B) HEK293 cells stably expressing tagged Nrf1 (Nrf13xFLAG) were treated or not with 200 nM CFZ for 8 hours. The cell lysates were then subjected to immunoprecipitation with anti-FLAG beads and analyzed by immunoblotting with antibodies specific for FLAG, RUVBL1, and RUVBL2. The lysate lanes were loaded with 5% of the input that was used for immunoprecipitation. The experiments were performed three independent times and a representative blot is shown.

Article Snippet: The antibodies used were specific for Nrf1 (1:5000), RUVBL1 (1:2500), RUVBL2 (1:2500), Ubiquitin (1:3000), cleaved caspase-3 (1:3000) (all from Cell Signaling), INO80 (1:500; a gift from Dr. Landry (45)), PIH1 (1:1000), (Proteintech) and TIP60 (1:1500) (Abcam) and -Actin (1:10,000) (SigmaAldrich).

Techniques: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Immunoprecipitation, Western Blot

Journal: Chromosoma

Article Title: COORDINATION OF THE RECRUITMENT OF THE FANCD2 AND PALB2 FANCONI ANEMIA PROTEINS BY A UBIQUITIN SIGNALING NETWORK

doi: 10.1007/s00412-016-0602-9

Figure Lengend Snippet: a Representative immunoblot showing knockdown of MDC1. b–c Representative images (b) and quantification (c) of FANCD2 focus formation in HeLa cells transfected with a control siRNA or two distinct siRNAs targeting MDC1 and treated with 0.5 µM MMC for 16 hr. FANCD2 foci (red) are also shown in merged images in b with DAPI signal in blue to indicate the position of nuclei. d–e Representative images of MMC-induced PALB2 foci (d) in cells transfected with either siLacZ or MDC1 siRNAs and quantification (e) of the percentage of HeLa cells with five or more PALB2 foci. PALB2 foci (red) are also shown in merged images in d with DAPI signal in blue. f Immunoblot showing FANCD2 monoubiquitination status in cells treated with a siRNA directed against RNF8 or MDC1. The intensity of the upper monoubiquitinated band divided by the intensity of the lower unubiquitinated band is shown for each lane. g Quantification of the percentage of HeLa cells with five or more ubiquitin foci following transfection with siRNAs and treatment with MMC. Values in c,e,g represent the mean of three independent counts of at least 150 cells each ± standard deviation; * indicates p<0.005.

Article Snippet: Antibodies Primary antibodies utilized for immunofluorescence microscopy and immunoblotting were as follows: FK2 (EMD Millipore, 04-263), FANCD2 (E35) ( Garcia-Higuera et al. 2001 ), PALB2 ( Zhang et al. 2009a ), γH2AX (EMDMillipore, JBW301), RNF8 (Santa Cruz, sc271462), MDC1 (Novus Biologicals, NB100-395), FAAP20 ( Ali et al. 2012 ), RAP80 (Bethyl Laboratory, A300-763A), and anti-HA (Covance, 16B12).

Techniques: Western Blot, Knockdown, Transfection, Control, Ubiquitin Proteomics, Standard Deviation

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting the model system employed to visualise TIGIT on the surface of T cells when interacting with Raji B cells expressing different nectin ligands. b Flow cytometry analysis showing the expression of TIGIT in Jurkat cells (above) and CD111 and CD155 in Raji cells (below), in both the parental and expression lines together with isotype-matched controls. c Confocal microscopy images showing TIGIT-GFP (green) on the surface of Jurkat cells (T) conjugated for 20 mins with different Raji cell (B) populations, as indicated to the left of the panel. CD19 (yellow) is used to mark Raji cells and a V5 stain labels expressed nectins (magenta). Respective brightfield images (BF) are also provided. The bottom two rows show Jurkat T cells that have been preincubated with either an antagonistic TIGIT antibody or an isotype-matched control. d Mean log 2 fold change in synaptic TIGIT enrichment in Jurkat cells, from the conjugates shown in c (±S.D.; n = 3 independent experiments; adjusted P values from a one-way ANOVA with Tukey’s multiple comparisons are given; ns = not significant). e Representative confocal microscopy images showing TIGIT (green) on the surface of primary T cells conjugated with different Raji B cell populations, as indicated to the left. CD4 and CD8 (yellow) were stained to mark T cell subsets, and BF provided. f Mean log 2 fold change (±S.D., n = 3 independent donors matched by colour) in synaptic TIGIT enrichment in primary T cells, from the conjugates shown in e . Adjusted P values from a paired T-test are given (Holm-Šídák method). g Schematic depicting the model system employed to test the inhibitory effect of TIGIT on the surface of Jurkat T cells when interacting with cells expressing different nectin ligands. Staphylococcal Enterotoxin E (SEE) was used to stimulate Jurkat cells. h Relative amounts of IL-2 released from either parental or TIGIT-SNAP-expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells for 6 h. Data is shown as the mean log 2 fold changes between Raji-CD155 conjugates compared to Raji-CD111 conjugates (±S.D., n = 5 independent experiments with adjusted P values from a one-way ANOVA with Holm-Šídák’s multiple comparisons displayed). Cells pre-incubated with an antagonistic TIGIT antibody (αT) or an isotype-matched control (iso) are shown, as indicated. All scale bars = 5 µm. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence experiments the following antibodies were used: αTIGIT (MBSA43; 2.5 μg/mL), GFP-Booster nanobodies (Atto488-, Alexa Fluor 488- and Alexa Fluor 647-conjugated; gba488, gb2AF488 and gb2AF647; ChromoTek; 1 μg/mL), αDNAM-1 (Clone DX11; 2.5 μg/mL), αCD96 (Clone NK92.39; 5 μg/mL), αV5 (Rabbit polyclonal; NB600-381; Novus Biologicals; 1 μg/mL), αCD19 (Clone HIB19; 302250; BioLegend; 2 μg/mL), αCD4 (Clone MT310; sc-19641; Santa Cruz Biotechnology; 2 μg/mL) and αCD8 (Clone RPA-T8; 301062; 2.5 μg/mL).

Techniques: Expressing, Flow Cytometry, Confocal Microscopy, Staining, Control, Incubation

Journal: Nature Communications

Article Title: TIGIT can inhibit T cell activation via ligation-induced nanoclusters, independent of CD226 co-stimulation

doi: 10.1038/s41467-023-40755-3

Figure Lengend Snippet: a Schematic depicting individual point mutations introduced into TIGIT-SNAP. b Representative confocal microscopy images showing WT and mutant forms of TIGIT-SNAP (green) on the surface of Jurkat T cells conjugated for 20 mins with different Raji B cell populations (either CD111- or CD155-expressing; stained via V5 and shown in magenta). A merged fluorescence-BF image is also provided. c Mean log 2 fold change (±S.D., n = 3 independent experiments) in synaptic TIGIT enrichment in Jurkat T cells, from the conjugates shown in b . Adjusted P values from a one-way ANOVA with Šídák’s multiple comparisons are displayed, with differences from the WT-111 condition displayed in black and the WT-155 condition displayed in grey. d Representative TIRF microscopy images of WT and mutant forms of TIGIT-SNAP at the IS of Jurkat cells that have interacted with PLBs loaded with ICAM-1, and CD111 or CD155 for 20 mins, as in Fig. . Intensities have been scaled equally, and colour scales provided. e Mean degree of TIGIT clustering measured from the images shown in d (±S.D., n = 3–4 independent experiments, as indicated). Adjusted P values from a one-way ANOVA with Dunnett’s multiple comparisons are displayed, and coloured as in c . f ELISA data showing the relative amount of IL-2 released from either parental or different forms of TIGIT-SNAP-expressing Jurkat cells after co-incubation with SEE-pulsed Raji cells. Data is shown as the mean log 2 fold changes between Raji-CD155 conjugates compared to Raji-CD111 conjugates, ± S.D. ( n = ≧5 independent experiments with adjusted P values from a one-way mixed-effects analysis with a Dunnett’s multiple comparison test displayed). Differences from the parental condition are displayed above in black and from the WT condition displayed below in grey. g Western blot analysis of TIGIT using either Phos-tag SDS-PAGE (left) or standard SDS-PAGE to examine TIGIT phosphorylation in Raji-Jurkat conjugates, as labelled above. Data are representative of 3 independent experiments. h Representative TIRF microscopy images of different forms of TIGIT (SNAP labelled; magenta) and the TCR (OKT3 in PLB; green) in Jurkat cells upon interaction with PLBs containing ICAM-1, either CD111 or CD155, and fluorescently labelled OKT3 (100 molecules/μm 2 ), for 10 mins. i Mean Pearson’s correlation coefficient (±S.D; n = ≧50 cells from 2 independent experiments) between TIGIT and OKT3 from the images shown in h . Adjusted P values from a Kruskal-Wallis test with Dunn’s multiple comparisons are shown, and coloured as in c . All scale bars = 5 μm. Source data are provided as a Source Data file.

Article Snippet: For immunofluorescence experiments the following antibodies were used: αTIGIT (MBSA43; 2.5 μg/mL), GFP-Booster nanobodies (Atto488-, Alexa Fluor 488- and Alexa Fluor 647-conjugated; gba488, gb2AF488 and gb2AF647; ChromoTek; 1 μg/mL), αDNAM-1 (Clone DX11; 2.5 μg/mL), αCD96 (Clone NK92.39; 5 μg/mL), αV5 (Rabbit polyclonal; NB600-381; Novus Biologicals; 1 μg/mL), αCD19 (Clone HIB19; 302250; BioLegend; 2 μg/mL), αCD4 (Clone MT310; sc-19641; Santa Cruz Biotechnology; 2 μg/mL) and αCD8 (Clone RPA-T8; 301062; 2.5 μg/mL).

Techniques: Confocal Microscopy, Mutagenesis, Expressing, Staining, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Incubation, Comparison, Western Blot, SDS Page, Phospho-proteomics

Journal: Cell stem cell

Article Title: Selective translation of cell fate regulators mediates tolerance to broad oncogenic stress

doi: 10.1016/j.stem.2020.05.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used: mouse anti-eIF2B5 (B-7, 1:50; Santa Cruz Biotechnology); mouse anti-β-Actin (2D4H5, 1:3000 for WB; Proteintech); mouse anti-Puromycin (12D10, 1:1000; Millipore); mouse anti-EIF2α (L57A5, 1:500; Cell Signaling); rabbit anti-phospho-EIF2α Ser51 (119A11, 1:500; Cell Signaling); rabbit anti-FBXO32 (EPR9148(2), 1:500; Abcam); mouse anti-V5 (V5-10, 1:3000 for WB; Sigma-Aldrich).

Techniques: shRNA, Recombinant, Blocking Assay, Plasmid Preparation, Lysis, SYBR Green Assay, Imaging, Activity Assay, Mutagenesis, Bicinchoninic Acid Protein Assay, Reporter Assay, Software