anti-p-eif2α (s51) (119a11)  (Cell Signaling Technology Inc)

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Immunohistochemical (IHC) staining for p-eIF2α S51 of a tissue microarray from colon mucosa ( n = 84), adenoma ( n = 34) and colon carcinoma ( n = 54). The IHC pictures are representative of the respective samples. Scale bar = 100 µM. ( B ) Quantification of p-eIF2α S51 IHC described in ( A ). Staining intensity scores are displayed; 0 = negative, 1 = weak, 2 = intermediate, 3 = strong. Above each stage, the mean of p-eIF2α S51 staining intensity is given. ( C ) Western blot of indicated proteins in murine WT, A, AK, and LAKTP intestinal organoids, representative of three biological replicates with similar results. Levels of p-eIF2α S51, relative to total eIF2α and normalized to vinculin, are given below the blot. ( D ) Protein synthesis in murine WT, A, AK, and LAKTP intestinal organoids analyzed by puromycin incorporation. As control, organoids were treated with cycloheximide (50 µg/ml) to inhibit protein synthesis. Western blot is representative of two biological replicates with similar results. Puromycin incorporation, relative to vinculin, is shown below. ( E ) Growth curve of SW480 cells treated with indicated concentrations of ISRIB or DMSO as control for 4 days. Cell number was analyzed with the Operetta screening microscope. Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( F ) Pictures of A, AK, LAKTP murine intestinal organoids and T4, FAP, HD-3 PDOs treated with indicated concentrations of ISRIB for 5 days, representative of three to four biological replicates with similar results. Scale bar = 200 µm. ( G ) Relative viability of A, AK, LAKTP murine intestinal organoids and T4, FAP, HD-3 PDOs treated as described in ( F ). Data show mean ± s.d. ( n = 3 or 4 biological replicates); Student’s t test. .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Immunohistochemical staining, Immunohistochemistry, Microarray, Staining, Western Blot, Control, Microscopy

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Western blot of indicated proteins in SW480 cells transduced with shCTRL or shRNAs against EIF2B1-4 , representative of three biological replicates with similar results. Levels of the respective eIF2B subunits, relative to vinculin, are given below each corresponding panel. ( B ) Protein synthesis in SW480 cells transduced as described in ( A ) analyzed by puromycin incorporation. As control, cells were treated with cycloheximide (50 µg/ml) to inhibit protein synthesis. The western blot is representative of three biological replicates with similar results. Levels of puromycin incorporation, relative to vinculin, are given below the blot. ( C ) Western blot of indicated proteins in SW480 cells transduced as described in ( A ), representative of three biological replicates with similar results. Levels of p-eIF2α S51, relative to total eIF2α and normalized to vinculin, are given below the blot. ( D ) Growth curve of SW480 cells transduced as described in ( A ), measured with Incucyte ® live-cell imaging system. Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( E ) Annexin V/PI FACS analysis of SW480 cells transduced as described in ( A ). Data show mean ± s.d. ( n = 3 biological replicates). Student’s t test. ( F ) PI cell cycle FACS analysis of SW480 cells transduced as described in ( A ). Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( G ) Length of G1 cell cycle phase of SW480 cells transduced as described in ( A ), calculated with data acquired from growth curve ( D ) and PI cell cycle FACS ( F ). Data show mean ± s.e.m. ( n = 3 biological replicates); Student’s t test. .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Western Blot, Transduction, Control, Live Cell Imaging

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Cryo-EM structure of the eIF2Bα homodimer (PDB: 6O81) with four amino acids of each eIF2Bα monomer, A181, Y185, E188 and C218, shown in detail. ( B ) Western blot of indicated proteins in shCTRL- or sh EIF2B1 -transduced SW480 cells stably overexpressing eIF2Bα mutant (eIF2Bα mut ), eIF2Bα WT (eIF2Bα wt ) construct, or without any overexpression (empty). The western blot is representative of three biological replicates with similar results. ( C ) mRNA expression of endogenous EIF2B1 in SW480 cells transduced as described in ( B ). Primers targeting the EIF2B1 3’ UTR were used. Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( D ) Western blot of indicated proteins in SW480 cells transduced as described in ( B ), representative of three biological replicates. ( E ) Quantification of p-eIF2α S51 levels and total eIF2α levels, normalized to vinculin, of western blots described in ( D ). Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( F ) Growth curve of SW480 cells transduced as described in ( B ) or treated with 1000 nM ISRIB for 7 days (DMSO as control), measured with Incucyte ® live-cell imaging system. Data show mean ± s.d. ( n = 6 biological replicates); Student’s t test. ( G ) Annexin V/PI FACS analysis of SW480 cells transduced as described in ( B ). Data show mean ± s.d. ( n = 4 biological replicates); Student’s t test. ( H ) Immunoprecipitation of eIF2α in SW480 cells stably overexpressing HA-tagged eIF2Bα wt or eIF2Bα mut . As input, 2% of lysate was loaded. Co-immunoprecipitated HA-eIF2Bα constructs were detected by western blot. The western blot is representative of four biological replicates with similar results. .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Cryo-EM Sample Prep, Western Blot, Stable Transfection, Mutagenesis, Construct, Over Expression, Expressing, Control, Live Cell Imaging, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Western blot of indicated proteins in shCTRL- or sh EIF2B1 -transduced SW480 cells stably overexpressing eIF2α WT (eIF2α wt ), eIF2α S51A mutant (eIF2α S51A ) construct, or without any overexpression (empty). The western blot is representative of three biological replicates with similar results. ( B ) Growth curve of SW480 cells transduced as described in ( A ), measured with Incucyte ® live-cell imaging system. Data show mean ± s.d. ( n = 4 biological replicates); Student’s t test. ( C ) Western blot of indicated proteins in shCTRL- or sh EIF2B1 -transduced SW480 cells stably overexpressing eIF2α S51D mutant (eIF2α S51D ) construct, or without any overexpression (empty). The western blot is representative of three biological replicates with similar results. ( D ) Growth curve of SW480 cells transduced as described in ( C ), measured with Incucyte ® live-cell imaging system. Data show mean ± s.d. ( n = 4 biological replicates); Student’s t test. ( E ) mRNA expression of PPP1R15A in shCTRL- or sh EIF2B1 -transduced SW480 cells stably overexpressing eIF2Bα mutant (eIF2Bα mut ), eIF2Bα WT (eIF2Bα wt ) construct, or without any overexpression (empty), transfected with siCTRL or si PPP1R15A for 72 hr. Data show mean of technical triplicates of one representative experiment ( n = 2 biological replicates). ( F ) Western blot of indicated proteins in SW480 cells transduced and transfected as described in ( E ). The western blot is representative of two biological replicates with similar results. Levels of p-eIF2α S51, relative to total eIF2α, are given below. ( G ) Crystal violet staining of SW480 cells transduced and transfected as described in ( E ). Staining was done 7 days after seeding. Pictures are representative of two biological replicates with similar results. ( H ) Quantification of crystal violet staining described in ( G ). Data show mean ( n = 2 biological replicates). .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Western Blot, Stable Transfection, Mutagenesis, Construct, Over Expression, Live Cell Imaging, Expressing, Transfection, Staining

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Cryo-EM structure of eIF2Bδ in complex with p-eIF2α (PDB: 6O9Z) with two amino acids of eIF2Bδ, A315 and A318, shown in detail. ( B ) Western blot of indicated proteins in shCTRL- or sh EIF2B4 -transduced SW480 cells stably overexpressing eIF2Bδ mutant (eIF2Bδ mut ), eIF2Bδ WT (eIF2Bδ wt ) construct, or without any overexpression (empty). The western blot is representative of three biological replicates with similar results. ( C ) mRNA expression of endogenous EIF2B4 in SW480 cells transduced as described in ( B ). Primers targeting the EIF2B4 3’ UTR were used. Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( D ) Western blot of indicated proteins in SW480 cells transduced as described in ( B ), representative of three biological replicates with similar results. ( E ) Quantification of p-eIF2α S51 levels and total eIF2α levels, normalized to vinculin, of western blots described in ( D ). Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( F ) Growth curve of SW480 cells transduced as described in ( B ), measured with Operetta screening microscope. Data show mean ± s.e.m. ( n = 3 biological replicates); Student’s t test. ( G ) Annexin V/PI FACS analysis of SW480 cells transduced as described in ( B ). Data show mean ± s.d. ( n = 3 biological replicates). Student’s t test. ( H ) Immunoprecipitation of eIF2α in SW480 cells stably overexpressing HA-tagged eIF2Bδ wt or eIF2Bδ mut . As input, 2% of lysate was loaded. Co-immunoprecipitated HA-eIF2Bδ constructs were detected by western blot. The western blot is representative of three biological replicates with similar results. .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Cryo-EM Sample Prep, Western Blot, Stable Transfection, Mutagenesis, Construct, Over Expression, Expressing, Microscopy, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Pictures of murine WT, A, AK and LAKTP intestinal organoids transduced with doxycycline-inducible shCTRL or sh Eif2b1-1 (7 days of doxycycline treatment), representative of three biological replicates with similar results. Green signal (GFP) indicates shRNA induction. Scale bar = 200 µm. ( B ) Relative viability of WT, A, AK, and LAKTP organoids transduced and treated as described in ( A ). Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( C ) Western blot of indicated proteins in WT, A, AK, and LAKTP organoids transduced as described in ( A ), representative of two or three biological replicates with similar results (96 h of doxycycline treatment); *unspecific bands. ( D ) Quantification of eIF2Bα, p-eIF2α S51 and total eIF2α levels, normalized to vinculin, of western blots described in ( C ). Data show mean ± s.d. of two to three biological replicates; Student’s t test. ( E ) Pictures of human WT Ko165 organoids and T4, FAP, HD-3 PDOs transduced with doxycycline-inducible shCTRL or sh EIF2B1-1 (7 days of doxycycline treatment), representative of three biological replicates with similar results. Green signal (GFP) indicates shRNA induction. Scale bar = 200 µm. ( F ) Relative viability of WT Ko165, T4, FAP, HD-3 organoids transduced and treated as described in ( E ). Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( G ) Western blot of indicated proteins in WT Ko165, T4, FAP, HD-3 organoids transduced as described in ( E ), representative of two or three biological replicates with similar results (96 h of doxycycline treatment). ( H ) Quantification of eIF2Bα, p-eIF2α S51 and total eIF2α levels, normalized to vinculin, of western blots described in ( G ). Data show mean ± s.d. ( n = 2 or 3 biological replicates); Student’s t test. .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Transduction, shRNA, Western Blot

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: ( A ) Pictures of murine A, AK and LAKTP intestinal organoids transduced with doxycycline-inducible shCTRL or sh Eif2b1-2 (7 days of doxycycline treatment), representative of five biological replicates with similar results. Green signal (GFP) indicates shRNA induction. Scale bar = 200 µm. ( B ) Relative viability of A, AK and LAKTP organoids transduced and treated as described in ( A ). Data show mean ± s.d. ( n = 5 biological replicates); Student’s t test. ( C ) Western blot of indicated proteins in A, AK and LAKTP organoids transduced as described in (A), representative of two or three biological replicates with similar results (96 h of doxycycline treatment); *unspecific bands. ( D ) Quantification of eIF2Bα, p-eIF2α S51 and total eIF2α levels, normalized to vinculin, of western blots described in ( C ). Data show mean ± s.d. ( n = 2 or 3 biological replicates); Student’s t test. ( E ) Pictures of human T4, FAP, HD-3 PDOs transduced with doxycycline-inducible shCTRL or sh EIF2B1-2 (7 days of doxycycline treatment), representative of three biological replicates with similar results. Green signal (GFP) indicates shRNA induction. Scale bar = 200 µm. ( F ) Relative viability of T4, FAP, HD-3 PDOs transduced and treated as described in ( E ). Data show mean ± s.d. ( n = 3 biological replicates); Student’s t test. ( G ) Western blot of indicated proteins in T4, FAP, HD-3 PDOs transduced as described in ( E ), representative of three or four biological replicates with similar results (96 h of doxycycline treatment). ( H ) Quantification of eIF2Bα, p-eIF2α S51 and total eIF2α levels, normalized to vinculin, of western blots described in ( G ). Data show mean ± s.d. ( n = 3 or 4 biological replicates); Student’s t test. .

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Transduction, shRNA, Western Blot

Journal: The EMBO Journal

Article Title: Maintenance of p-eIF2α levels by the eIF2B complex is vital for colorectal cancer

doi: 10.1038/s44318-025-00381-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: p-eIF2α S51 , Cell Signaling Technology , 3398.

Techniques: Recombinant, Sequencing, Protease Inhibitor, Viability Assay, Western Blot, SYBR Green Assay, Membrane, RNA Sequencing, Software, Imaging, Control, Real-time Polymerase Chain Reaction, High Content Screening

Journal: Journal of Biomedical Science

Article Title: The endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 3 attenuates the unfolded protein response and has pro-survival and pro-viral roles in hepatoma cells and hepatocellular carcinoma patients

doi: 10.1186/s12929-024-01103-9

Figure Lengend Snippet: UPR pathways are downregulated in EDEM3-overexpressing HepaRG cells. A EDEM3 expression in HepaRG C and HepaRG EDEM3 cells was determined by western blot. Detection of α-tubulin was used as total protein loading control. B Expression of EDEM3 ( green ) and PDI ( red ) was determined in HepaRG C and HepaRG EDEM3 cells by incubation with corresponding antibodies followed by immunofluorescence microscopy. Images were analyzed with the AxioVision SE64 Rel. 4.9.1 Software. The cell nuclei were stained with DAPI ( blue ). The indicated scale bar is 50 µm. C Expression of UPR and ERAD markers in HepaRG C and HepaRG EDEM3 cells was investigated by western blot with corresponding antibodies. Detection of α-tubulin, β-actin or calnexin was used as internal controls for total protein loading. The asterisk indicates a non-specific reactivity of the p - eIF2α antibody. The bands corresponding to the proteins of interest were quantified from three independent experiments by using the ImageJ software and normalized to corresponding internal controls. Statistical analysis of the relative protein expression using the unpaired t-test is shown (* p < 0.05, ** p < 0.01)

Article Snippet: Primary antibodies used for western blot analyses include mouse anti-EDEM3 (E0409, Sigma-Aldrich, 1:500), rabbit anti-EDEM1 (E8406, Sigma-Aldrich, 1:1000), rabbit anti-EDEM2 (JD-32) (sc - 130460, Santa Cruz Biotechnology, 1:250), rabbit anti-HBsAg (NB100-62652, Novus Biologicals, Littleton, CO, USA, 1:1000), rabbit anti-phospho-IRE1 alpha (Ser724) (PA1 - 16927, Invitrogen, 1:1000), rabbit anti-IRE1α (14C10, Cell Signaling Technology, Danvers, MA, USA, 1:1000), mouse anti - ATF6 (70B1413.1, Novus Biologicals, 1:1000), anti-glucose-regulated protein 94 (GRP94; H-10, Santa Cruz Biotechnology, 1:500), rabbit anti - phospho - PERK-T980 (16F8, Cell Signaling Technology, 1:1000), rabbit anti-PERK (C33E10, Cell Signaling Technology, 1:1000), rabbit anti - phospho-alpha subunit of eukaryotic initiation factor 2, S51(p - eIF2α; 9721S, Cell Signaling Technology, 1:1000), rabbit anti-eIF2α (9722S, Cell Signaling Technology, 1:1000), mouse anti-binding immunoglobulin protein (GRP78/BiP; A-10; sc-376768, Santa Cruz Biotechnology, 1:200), rabbit anti-suppressor of Lin - 12 - like (Sel1L; ab78298, Cell Signaling Technology, 1:500), rabbit anti-HMG-CoA reductase degradation protein 1, also known as Synoviolin (Hrd1; 14773, Cell Signaling Technology, 1:1000), rabbit anti-ER lectin 1 (XTP3-B/ERLEC1; ab181166, Abcam, Cambridge, UK, 1:500), rabbit anti-osteosarcoma amplified - 9 (OS-9; ab19853, Abcam, 1:500), rabbit anti-Ras homolog enriched in the brain (Rheb; E1G1R, Cell Signaling Technology, 1:1000), goat anti-apolipoprotein E (ApoE; AB947, Sigma-Aldrich, 1:5000), rabbit anti-microtubule-associated protein light chain 3A/B (LC3A/B; D3U4C, Cell Signaling Technology, 1:1000), rabbit anti-phospho-mammalian target of rapamycin (mTOR Ser2448; D9C2, Cell Signaling Technology, 1:1000), rabbit anti-mTOR (7C10, Cell Signaling Technology, 1:1000), mouse anti-p53 (DO-1, sc-126, Santa Cruz Biotechnology, 1:500), mouse anti-pro-apoptotic effector B-cell lymphoma protein 2 (Bcl-2) Associated X (BAX; MA5-14003, Invitrogen, 1:500), rabbit anti-calnexin (ab22595, Abcam, 1:5000), mouse anti - β - actin (ab8224, Abcam, 1:5000), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; PA1-987, Invitrogen, 1:5000), rabbit anti-α-tubulin (ab6046, Abcam, 1:10000).

Techniques: Expressing, Western Blot, Control, Incubation, Immunofluorescence, Microscopy, Software, Staining

Journal: Journal of Biomedical Science

Article Title: The endoplasmic reticulum degradation-enhancing α-mannosidase-like protein 3 attenuates the unfolded protein response and has pro-survival and pro-viral roles in hepatoma cells and hepatocellular carcinoma patients

doi: 10.1186/s12929-024-01103-9

Figure Lengend Snippet: EDEM3 depletion promotes apoptosis in HepaRG cells. A Expression of UPR markers in HepaRG and HepaRG EDEM3KO cells was determined by western blot with corresponding antibodies. Where indicated, cells were treated with 2.5 µg/mL TM for 6 h. Detection of calnexin was used as internal control. The asterisk indicates a non-specific reactivity of the p - eIF2α antibodies. The bands corresponding to the proteins of interest were quantified from three independent experiments by using the ImageJ software and normalized to corresponding internal controls. Statistical analysis of the relative protein expression using the unpaired t-test is shown (* p < 0.05, *** p < 0.001). B Cell apoptosis was evaluated by flow cytometry. Histograms indicate the percentage of viable and Annexin V+ cells, as detected on fluorescein isothiocyanate (FITC) channel. Data shown are representative of two independent experiments. C Expression of apoptotic markers in HepaRG and HepaRG EDEM3KO cells was determined by western blot with corresponding antibodies. Detection of calnexin was used as an internal control. The bands corresponding to the proteins of interest were quantified from three independent experiments by using the ImageJ software and normalized to corresponding internal controls. Statistical analysis of the relative protein expression using the unpaired t-test is shown (* p < 0.05). D Autophagy and mTOR activation markers were investigated in HepaRG and HepaRG EDEM3KO cells by western blot with corresponding antibodies. Where indicated, cells were treated with 2.5 µg/mL of TM for 6 h. Detection of calnexin or GAPDH was used as internal control. The bands corresponding to the proteins of interest were quantified from three independent experiments by using the ImageJ software and normalized to corresponding internal controls. Statistical analysis of the relative protein expression using the unpaired t-test is shown (* p < 0.05)

Article Snippet: Primary antibodies used for western blot analyses include mouse anti-EDEM3 (E0409, Sigma-Aldrich, 1:500), rabbit anti-EDEM1 (E8406, Sigma-Aldrich, 1:1000), rabbit anti-EDEM2 (JD-32) (sc - 130460, Santa Cruz Biotechnology, 1:250), rabbit anti-HBsAg (NB100-62652, Novus Biologicals, Littleton, CO, USA, 1:1000), rabbit anti-phospho-IRE1 alpha (Ser724) (PA1 - 16927, Invitrogen, 1:1000), rabbit anti-IRE1α (14C10, Cell Signaling Technology, Danvers, MA, USA, 1:1000), mouse anti - ATF6 (70B1413.1, Novus Biologicals, 1:1000), anti-glucose-regulated protein 94 (GRP94; H-10, Santa Cruz Biotechnology, 1:500), rabbit anti - phospho - PERK-T980 (16F8, Cell Signaling Technology, 1:1000), rabbit anti-PERK (C33E10, Cell Signaling Technology, 1:1000), rabbit anti - phospho-alpha subunit of eukaryotic initiation factor 2, S51(p - eIF2α; 9721S, Cell Signaling Technology, 1:1000), rabbit anti-eIF2α (9722S, Cell Signaling Technology, 1:1000), mouse anti-binding immunoglobulin protein (GRP78/BiP; A-10; sc-376768, Santa Cruz Biotechnology, 1:200), rabbit anti-suppressor of Lin - 12 - like (Sel1L; ab78298, Cell Signaling Technology, 1:500), rabbit anti-HMG-CoA reductase degradation protein 1, also known as Synoviolin (Hrd1; 14773, Cell Signaling Technology, 1:1000), rabbit anti-ER lectin 1 (XTP3-B/ERLEC1; ab181166, Abcam, Cambridge, UK, 1:500), rabbit anti-osteosarcoma amplified - 9 (OS-9; ab19853, Abcam, 1:500), rabbit anti-Ras homolog enriched in the brain (Rheb; E1G1R, Cell Signaling Technology, 1:1000), goat anti-apolipoprotein E (ApoE; AB947, Sigma-Aldrich, 1:5000), rabbit anti-microtubule-associated protein light chain 3A/B (LC3A/B; D3U4C, Cell Signaling Technology, 1:1000), rabbit anti-phospho-mammalian target of rapamycin (mTOR Ser2448; D9C2, Cell Signaling Technology, 1:1000), rabbit anti-mTOR (7C10, Cell Signaling Technology, 1:1000), mouse anti-p53 (DO-1, sc-126, Santa Cruz Biotechnology, 1:500), mouse anti-pro-apoptotic effector B-cell lymphoma protein 2 (Bcl-2) Associated X (BAX; MA5-14003, Invitrogen, 1:500), rabbit anti-calnexin (ab22595, Abcam, 1:5000), mouse anti - β - actin (ab8224, Abcam, 1:5000), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; PA1-987, Invitrogen, 1:5000), rabbit anti-α-tubulin (ab6046, Abcam, 1:10000).

Techniques: Expressing, Western Blot, Control, Software, Flow Cytometry, Activation Assay

Journal: EMBO Reports

Article Title: CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

doi: 10.1038/s44319-024-00248-w

Figure Lengend Snippet: ( A ) Immunoblotting and quantification of p-eIF2α (S51) in HEK293 and HeLa cells treated with indicated drugs (MG132 (1 μM), KN-93 (10 μM)) for 14 h. Data are mean ± SEM of biological replicates ( n = 3). ( B ) Representative p-eIF2α (S51) staining images of HeLa cells treated with indicated drugs (MG132 (1 μM), KN-93 (10 μM)) for 14 h. Scale bar: 20 μm. ( C ) Immunofluorescence-based measurement and quantification of the expression levels of p-eIF2α shown in ( B ). Data are mean ± SEM of biological replicates ( n = 3). ( D ) Immunoblotting and quantification of p-eIF2α (S51) in HEK293 and HeLa cells treated with indicated drugs (MG132 (1 μM), KN-93 (10 μM), BTdCPU (2 μM)) for 14 h. Data are mean ± SEM of biological replicates ( n = 4). ( E ) Immunoblotting and quantification of UB in NP-40-soluble and -insoluble fractions of HEK293 cells treated with indicated drugs for 14 h. Data are mean ± SEM of biological replicates ( n = 3). ( F ) Representative UB-K48 staining images of HeLa cells treated indicated drugs for 14 h. Scale bar: 20 μm. ( G ) Quantitative analysis of results in ( F ). Data are mean ± SEM of biological replicates ( n = 3). At least 50 cells were randomly selected from each group to score. ( H ) Cell morphology imaging of HEK293 and HeLa cells treated with indicated drugs for 24 h. Scale bar: 10 μm. ( I ) Quantitative analysis of results in ( H ). Data are mean ± SEM of biological replicates ( n = 3). At least 50 cells were randomly selected from each group to score. ( J ) Representative images of HeLa cells transfected with ER-EGFP and treated with indicated drugs for 24 h. Mitochondria and nuclei were stained with Mito-Tracker (red) and Hoechst (blue), respectively. Scale bar: 20 μm. ( K, L ) Cell viability of HEK293 and HeLa cells treated with indicated drugs (MG132 (1 μM), KN-93 (10 μM), BTdCPU (0.5 μM, 1 μM, 2 μM)) for 48 h. Data are mean ± SEM of biological replicates ( n = 3). For ( A , C , D , E , G , I , K , L ), the P value was determined by a one-way ANOVA analysis. NS not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. .

Article Snippet: Rabbit anti-p-eIF2α S51 , Proteintech , Cat#28740-1-AP.

Techniques: Western Blot, Staining, Immunofluorescence, Expressing, Imaging, Transfection

Journal: EMBO Reports

Article Title: CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

doi: 10.1038/s44319-024-00248-w

Figure Lengend Snippet: ( A ) Confirmation of EIF2AKs knockdown in HEK293 and HeLa cells. After transfected with indicated siRNAs, the whole cell lysates were subjected to western blot analysis with indicated antibodies. ( B ) Quantitative analysis of results in ( A ). Data are mean ± SEM of biological replicates ( n = 3). ( C ) Immunoblotting and quantification of p-eIF2α (S51) in HeLa cells transfected with indicated siRNAs and treated with MG132. Data are mean ± SEM of biological replicates ( n = 3). ( D ) Immunoblotting and quantification of UB in NP-40-soluble and -insoluble fractions of HEK293 and HeLa cells transfected with indicated siRNAs and treated with MG132. Data are mean ± SEM of biological replicates ( n = 3). ( E ) Cell morphology imaging of HEK293 and HeLa cells transfected with indicated siRNAs and treated with MG132. Scale bar: 10 μm. ( F ) Quantitative analysis of results in ( E ). Data are mean ± SEM of biological replicates ( n = 3). At least 50 cells were randomly selected from each group to score. ( G ) Cell morphology imaging of HEK293 and HeLa cells treated with indicated drugs (Bortezomib (1 μM), KN-93 (10 μM), BTdCPU (0.5 μM)) for 24 h. Scale bar: 10 μm. ( H ) Quantitative analysis of results in ( G ). Data are mean ± SEM of biological replicates ( n = 3). At least 50 cells were randomly selected from each group to score. For ( B ), the P value was determined by Student’s t test (two-sided). ** P < 0.01; *** P < 0.001. For ( C , D , F , H ), the P value was determined by a one-way ANOVA analysis. NS not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Rabbit anti-p-eIF2α S51 , Proteintech , Cat#28740-1-AP.

Techniques: Knockdown, Transfection, Western Blot, Imaging

Journal: EMBO Reports

Article Title: CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

doi: 10.1038/s44319-024-00248-w

Figure Lengend Snippet: ( A , B ) Immunoblotting and quantification of p-eIF2α (S51) in BAG3 knockout cells treated with 1 μM MG132 for 14 h. Data are mean ± SEM of biological replicates ( n = 3). The P value was determined by Student’s t test (two-sided). NS not significant; * P < 0.05; ** P < 0.01. ( C – G ) Mass spectrum of the phosphopeptide containing the S173/S377/S386 phosphorylated residue of BAG3. ( H , I ) Phosphorylation level of re-expressed BAG3 (WT or mutants) in BAG3 −/− HEK293 cells treated with MG132 ( I ) or not ( H ). ( J ) Confirmation of purified BAG3-Twin Strep (WT or 3A) by coomassie blue staining.

Article Snippet: Rabbit anti-p-eIF2α S51 , Proteintech , Cat#28740-1-AP.

Techniques: Western Blot, Knock-Out, Phospho-proteomics, Residue, Purification, Staining

Journal: EMBO Reports

Article Title: CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

doi: 10.1038/s44319-024-00248-w

Figure Lengend Snippet: ( A ) Interaction between endogenous BAG3 and HRI in HEK293 cells. ( B ) Representative HRI and FLAG staining images of HeLa cells that were transfected with BAG3-FLAG and treated with indicated drugs. Scale bar: 10 μm. The fluorescence intensity line is tracing corresponding to a white line. ( C ) The effect of kinase dead mutant of CaMKIIβ on the interaction between BAG3 and HRI. After transfected, HEK293 cells were treated with MG132, and immunoprecipitated using anti-FLAG antibody. ( D ) Interaction between BAG3 and HRI in BAG3 −/− HEK293 cells that were transiently re-expressed wild-type or mutated BAG3 and then treated with MG132. Immunoprecipitation was performed using anti-FLAG antibody. Quantitative analysis of results is showed as the ratio of co-immunoprecipitated HRI to BAG3-FLAG. Data are mean ± SEM of biological replicates ( n = 3). ( E ) Phosphorylation level of HRI in HEK293 cells transfected with HRI-FLAG and treated with indicated drugs. Data are mean ± SEM of biological replicates ( n = 3). ( F ) Phosphorylation level of HRI in BAG3 −/− HEK293 cells that were co-transfected with Myc-tagged HRI and wild-type or mutated BAG3 and then treated with MG132 or not. Data are mean ± SEM of biological replicates ( n = 3). ( G – H ) Immunoblotting and quantification of p-eIF2α (S51) in BAG3 −/− HEK293 and BAG3 −/− HeLa cells that were transiently re-expressed mutated BAG3 and then treated with MG132. Data are mean ± SEM of biological replicates ( n = 3). ( I ) Protein synthesis was detected using a Click-iT HPG method in BAG3 -/- HEK293 and BAG3 −/− HeLa cells transiently re-expressed wild-type or mutated BAG3 and then treated with MG132. Data are mean ± SEM of biological replicates ( n = 3). For ( E , I ), the P value was determined by a one-way ANOVA analysis. NS not significant; * P < 0.05; ** P < 0.01; *** P < 0.001. For ( D , F – H ), the P value was determined by Student’s t test (two-sided). NS not significant; * P < 0.05. .

Article Snippet: Rabbit anti-p-eIF2α S51 , Proteintech , Cat#28740-1-AP.

Techniques: Staining, Transfection, Fluorescence, Mutagenesis, Immunoprecipitation, Phospho-proteomics, Western Blot

Journal: EMBO Reports

Article Title: CaMKII suppresses proteotoxicity by phosphorylating BAG3 in response to proteasomal dysfunction

doi: 10.1038/s44319-024-00248-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Rabbit anti-p-eIF2α S51 , Proteintech , Cat#28740-1-AP.

Techniques: Recombinant, Sequencing, Amplification, Protease Inhibitor, Software, Virus, Cloning, Isolation, cDNA Synthesis, SYBR Green Assay, Magnetic Beads