Western Blot:Article Title: Multi-omics after O-GlcNAc alteration identified cellular processes promoting aneuploidy after loss of O-GlcNAc transferase.
Article Snippet: .. The following antibodies were used for immunoblotting: OGT (AL-34) and OGA (345) were generously provided by Dr. Gerald Hart (Department of Biochemistry and Molecular Biology, the University of Georgia); anti-O-linked N-acetylglucosamine antibody (RL2, Thermo Fisher, MA1-072); anti-Actin antibody (Sigma Aldrich, A2066); Survivin (6E4, Cell Signaling, 2802S); p-Aurora A/B/C (T288, T232, T198) (D13A11, Cell Signaling, 2914P); monoclonal anti-AURKB antibody (Sigma Aldrich, WH0009212M3); p-cdc2 (Tyr15) (10A11, Cell Signaling, 4539S); anti-CDK1 antibody (A17, Abcam, ab18); anti-BUB1 antibody (EPR18947, Abcam, ab195268); anti-Cyclin B1 antibody (Sigma Aldrich, C8831); anti-Cyclin D1 (Sigma Aldrich, SAB4502602); phospho-YAP antibody (Ser127) (Cell Signaling, 4911); YAP (D8H1X, Cell Signaling, 14074); phospho-LATS1 (Ser909) (Cell Signaling, 9157); LATS1 (C55B5, Cell Signaling, 3477; Phospho-p70 S6 Kinase (Thr389) (Cell signaling #9205); p70 S6 Kinase Antibody (Cell signaling #9202). .. OSBP (Proteintech #11096-1-AP); MDM2 (Proteintech #27883-1-AP); AMPK-alpha (Cell signaling #5831S); p-AMPK (T172) (Cell signaling #2535S); p53 (Santa Cruz #G3014); p21 (Santa Cruz #A2413); ACC1(Cell signaling #4190S); p.ACC1 (Ser79) (Cell signaling#11818S); phospho-S6 (S235/236) (Cell signaling#4858S); S6 (S235/236) (Cell signaling#2217S); mTOR (Cell signaling#2972S); GAPDH (abcam #ab9485); goat anti-rabbit IgG-HRP (Bio-Rad, 170- 6515; goat anti-mouse IgG-HRP (Bio Rad, 170-6516); and anti-chicken IgY-HRP (Sigma Aldrich, A9046).
Article Title: Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for HCC
Article Snippet: .. CERS5 Rabbit mAb #37697 (SAB), LPCAT1 Rabbit mAb (ab214034), EGF Receptor (D38B1) XP Rabbit mAb #4267 (Cell Signaling Technology, CST), p44/42 mitogen-activated protein kinase (Erk1/2) (137F5) rabbit mAb #4695 (CST), Phospho-p44/42 mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (CST), beta-actin mouse mAb #60008-1-Ig (Proteintech), vinculin polyclonal antibody # 66305-1-Ig (E1E9V) (CST), Beclin1 mAb #66665-1-Ig (Proteintech), p70 S6 kinase antibody #9202 (CST), phospho-p70 S6 kinase (Thr389) Antibody #9205 (CST), and microtubule-associated protein 1 light chain 3 MAP1LC3 (LC3B) polyclonal antibody #14600-1-AP (Proteintech) were used for immunoblotting. ..
Article Title: Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for hepatocellular carcinoma
Article Snippet: .. [15] CERS5 Rabbit mAb #37697 (SAB), LPCAT1 Rabbit mAb (ab214034), EGF Receptor (D38B1) XP Rabbit mAb #4267 (Cell Signaling Technology, CST), p44/42 mitogen-activated protein kinase (Erk1/2) (137F5) rabbit mAb #4695 (CST), Phospho-p44/42 mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (CST), beta-actin mouse mAb #60008-1-Ig (Proteintech), vinculin polyclonal antibody # 66305-1-Ig (E1E9V) (CST), Beclin1 mAb #66665-1-Ig (Proteintech), p70 S6 kinase antibody #9202 (CST), phospho-p70 S6 kinase (Thr389) Antibody #9205 (CST), and microtubule-associated protein 1 light chain 3 MAP1LC3 (LC3B) polyclonal antibody #14600-1-AP (Proteintech) were used for immunoblotting. shRNA and transfection Short hairpin RNA (shRNA) stably knocked down targeted genes were established by lentiviral infection in cell lines. .. HEK293T cells were transfected with the shRNA vector and packaging plasmid mix using PEI DNA transfection reagents (# PR40001, Proteintech).
Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation
Article Snippet: .. GAPDH (glyceraldehyde-3-phosphate dehydrogenase; mouse, Sigma-Aldrich, G8795, 1:4000); actin ( mouse, Sigma-Aldrich, Cat# A5441, 1:4000);Phospho-p70 S6 Kinase (Thr389) (rabbit, Cell signaling, Cat# 9205L,1:500), p70 S6 Kinase Antibody (rabbit, Cell Signaling, 9202L, 1:1000); PI4K2A (homemade); MTMR14 (homemade); dsRed/mScarlet (rabbit, Clontech, Cat# 632496, 1:1000); pULK(S757) (rabbit, Cell signaling, Cat# 6888, 1:1000); ULK1(rabbit, Cell signaling, Cat# 8054, 1:1000); p4E-BP1(S65) (rabbit, Cell signaling, Cat# 13443; 1:500); 4EBP1 (rabbit, Cell signaling, Cat# 9644; 1:500); pTFEB(S211) (rabbit, Cell signaling, Cat# 37681, 1:1000); TFEB (rabbit, Biomol, Cat# A303-673A, 1:1000); Anti-mouse HRP (Jackson, Cat# 115-035-003, 1:4000); Anti-rabbit HRP (Jackson, Cat# 111-035-003, 1:4000); Anti-mouse Alexa568 (IgM-specific) (Thermo, Cat# A21043, immunofluorescence 1:400); PtdIns(4)P (Echelon, Cat# z-p004, immunofluorescence 1:200); a-RXRXX[S/T] (Cell Signaling, Cat# 10001, 1:500); pS6 (Cell Signaling, Cat# 2215, 1:1000); pS6(S240/244) (Cell Signaling, Cat# 5364, 1:1000); puromycin (Kerafast, Cat# Kf-Ab02366-23.0, WB 1:500, immunofluorescence 1:2000); CHMP4B (Proteintech, 13683-1-AP, immunofluorescence 1:200); IST1 (proteintech, Cat# 19842-1-AP, immunofluorescence 1:200); Alix (Biolegend, Cat# 634501); PSMB5 (Thermo, Cat# PA1-977, immunofluorescence 1:200); Anti-mouse CF568 (Biotium, Cat# 20109-0.25ml, immunofluorescence 1:1000); Anti-rabbit Alexa 647 (Thermo, Cat# A31573, immunofluorescence 1:1000); Anti-rabbit Alexa 568 (Thermo/ Molecular Probes, Cat# A11036, immunofluorescence 1:1000); Anti-mouse Alexa 647 (life, Cat# A21238, immunofluorescence 1:1000); anti-Mouse IRDye 680 CW (LICOR, P/N 925-68070, 1:4000); anti-Rabbit IRDye 680 CW (LICOR, P/N 925-68180, 1:4000). .. L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).
Article Title: PIM kinase control of CD8 T cell protein synthesis and cell trafficking
Article Snippet: Antibody , anti-rabbit IgG Fab2 Alexa Fluor 647 (goat) , Cell Signaling Technology , Cat # 4414 S, RRID: AB_10693544 , intracellular stain 1:1000. .. Antibody , p70 S6 Kinase Antibody (Rabbit, polyclonal) , Cell Signaling Technology , Cat # 9202 S, RRID: AB_331676 , western blot 1:1000. .. Antibody , Phospho-p70 S6 Kinase (Thr389) (Rabbit, monoclonal, 108D2) , Cell Signaling Technology , Cat # 9234 S, RRID: AB_2269803 , western blot 1:1000.
Article Title: Multi-omics after O-GlcNAc alteration identified cellular processes promoting aneuploidy after loss of O-GlcNAc transferase
Article Snippet: .. The following antibodies were used for immunoblotting: OGT (AL-34) and OGA (345) were generously provided by Dr. Gerald Hart (Department of Biochemistry and Molecular Biology, the University of Georgia); anti-O-linked N-acetylglucosamine antibody (RL2, Thermo Fisher, MA1-072); anti-Actin antibody (Sigma Aldrich, A2066); Survivin (6E4, Cell Signaling, 2802S); p-Aurora A/B/C (T288, T232, T198) (D13A11, Cell Signaling, 2914P); monoclonal anti-AURKB antibody (Sigma Aldrich, WH0009212M3); p-cdc2 (Tyr15) (10A11, Cell Signaling, 4539S); anti-CDK1 antibody (A17, Abcam, ab18); anti-BUB1 antibody (EPR18947, Abcam, ab195268); anti-Cyclin B1 antibody (Sigma Aldrich, C8831); anti-Cyclin D1 (Sigma Aldrich, SAB4502602); phospho-YAP antibody (Ser127) (Cell Signaling, 4911); YAP (D8H1X, Cell Signaling, 14074); phospho-LATS1 (Ser909) (Cell Signaling, 9157); LATS1 (C55B5, Cell Signaling, 3477; Phospho-p70 S6 Kinase (Thr389) (Cell signaling #9205); p70 S6 Kinase Antibody (Cell signaling #9202). .. OSBP (Proteintech #11096-1-AP); MDM2 (Proteintech #27883-1-AP); AMPK-alpha (Cell signaling #5831S); p-AMPK (T172) (Cell signaling #2535S); p53 (Santa Cruz #G3014); p21 (Santa Cruz #A2413); ACC1(Cell signaling #4190S); p.ACC1 (Ser79) (Cell signaling#11818S); phospho-S6 (S235/236) (Cell signaling#4858S); S6 (S235/236) (Cell signaling#2217S); mTOR (Cell signaling#2972S); GAPDH (abcam #ab9485); goat anti-rabbit IgG-HRP (Bio-Rad, 170-6515; goat anti-mouse IgG-HRP (Bio Rad, 170-6516); and anti-chicken IgY-HRP (Sigma Aldrich, A9046).
Article Title: STAUFEN1 regulating agents and associated methods
Article Snippet: .. The antibodies used for western blotting and their dilutions were as follows: mouse anti-Ataxin-2 antibody (Clone 22/Ataxin-2) [(1:4000), BD Biosciences, Cat #611378], rabbit anti-Staufen antibody [(1:5000), Novus biologicals, NBP1-33202], LC3B Antibody [(1:8000), Novus biologicals, NB100-2220], TDP-43 antibody [(1:8000), Proteintech, Cat #10782-2-AP], monoclonal anti-FLAG M2 antibody [(1:10,000), Sigma-Aldrich, F3165], monoclonal anti-β-Actin-peroxidase antibody (clone AC-15) [(1:30,000), Sigma-Aldrich, A3854], SQSTM1/p62 antibody [(1:4000), Cell Signaling, Cat #5114], mTOR antibody [(1:4000), Cell Signaling, Cat #2972], Phospho-mTOR (Ser2448) antibody [(1:3000), Cell Signaling, Cat #2971], p70 S6 Kinase antibody [(1:4000), Cell Signaling, Cat #9202], Phospho-p70 S6 Kinase (Thr389) antibody [(1:3000), Cell Signaling, Cat #9205], 4E-BP1 antibody [(1:4000), Cell Signaling, Cat #9452], Phospho-4E-BP1 (Thr37/46) (236B4) rabbit mAb [(1:3000), Cell Signaling, Cat #2855], 6×-His Tag monoclonal antibody (HIS.H8), HRP [(1:10,000) (ThermoFisher Scientific, MA1-21315-HRP)] and sheep-anti-Digoxigenin-POD, Fab fragments [(1:10,000), Roche Life Science, Cat #11207733910]. .. The secondary antibodies were: Peroxidase-conjugated horse anti-mouse IgG (H+L) antibody [(1:5000), Vector laboratories, PI-2000] and Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L) antibody [(1:5000), Jackson ImmunoResearch Laboratories, Cat #111-035-144].
shRNA:Article Title: Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for hepatocellular carcinoma
Article Snippet: .. [15] CERS5 Rabbit mAb #37697 (SAB), LPCAT1 Rabbit mAb (ab214034), EGF Receptor (D38B1) XP Rabbit mAb #4267 (Cell Signaling Technology, CST), p44/42 mitogen-activated protein kinase (Erk1/2) (137F5) rabbit mAb #4695 (CST), Phospho-p44/42 mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (CST), beta-actin mouse mAb #60008-1-Ig (Proteintech), vinculin polyclonal antibody # 66305-1-Ig (E1E9V) (CST), Beclin1 mAb #66665-1-Ig (Proteintech), p70 S6 kinase antibody #9202 (CST), phospho-p70 S6 kinase (Thr389) Antibody #9205 (CST), and microtubule-associated protein 1 light chain 3 MAP1LC3 (LC3B) polyclonal antibody #14600-1-AP (Proteintech) were used for immunoblotting. shRNA and transfection Short hairpin RNA (shRNA) stably knocked down targeted genes were established by lentiviral infection in cell lines. .. HEK293T cells were transfected with the shRNA vector and packaging plasmid mix using PEI DNA transfection reagents (# PR40001, Proteintech).
Transfection:Article Title: Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for hepatocellular carcinoma
Article Snippet: .. [15] CERS5 Rabbit mAb #37697 (SAB), LPCAT1 Rabbit mAb (ab214034), EGF Receptor (D38B1) XP Rabbit mAb #4267 (Cell Signaling Technology, CST), p44/42 mitogen-activated protein kinase (Erk1/2) (137F5) rabbit mAb #4695 (CST), Phospho-p44/42 mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (CST), beta-actin mouse mAb #60008-1-Ig (Proteintech), vinculin polyclonal antibody # 66305-1-Ig (E1E9V) (CST), Beclin1 mAb #66665-1-Ig (Proteintech), p70 S6 kinase antibody #9202 (CST), phospho-p70 S6 kinase (Thr389) Antibody #9205 (CST), and microtubule-associated protein 1 light chain 3 MAP1LC3 (LC3B) polyclonal antibody #14600-1-AP (Proteintech) were used for immunoblotting. shRNA and transfection Short hairpin RNA (shRNA) stably knocked down targeted genes were established by lentiviral infection in cell lines. .. HEK293T cells were transfected with the shRNA vector and packaging plasmid mix using PEI DNA transfection reagents (# PR40001, Proteintech).
Stable Transfection:Article Title: Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for hepatocellular carcinoma
Article Snippet: .. [15] CERS5 Rabbit mAb #37697 (SAB), LPCAT1 Rabbit mAb (ab214034), EGF Receptor (D38B1) XP Rabbit mAb #4267 (Cell Signaling Technology, CST), p44/42 mitogen-activated protein kinase (Erk1/2) (137F5) rabbit mAb #4695 (CST), Phospho-p44/42 mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (CST), beta-actin mouse mAb #60008-1-Ig (Proteintech), vinculin polyclonal antibody # 66305-1-Ig (E1E9V) (CST), Beclin1 mAb #66665-1-Ig (Proteintech), p70 S6 kinase antibody #9202 (CST), phospho-p70 S6 kinase (Thr389) Antibody #9205 (CST), and microtubule-associated protein 1 light chain 3 MAP1LC3 (LC3B) polyclonal antibody #14600-1-AP (Proteintech) were used for immunoblotting. shRNA and transfection Short hairpin RNA (shRNA) stably knocked down targeted genes were established by lentiviral infection in cell lines. .. HEK293T cells were transfected with the shRNA vector and packaging plasmid mix using PEI DNA transfection reagents (# PR40001, Proteintech).
Infection:Article Title: Comprehensive profiling of lipid metabolic reprogramming expands precision medicine for hepatocellular carcinoma
Article Snippet: .. [15] CERS5 Rabbit mAb #37697 (SAB), LPCAT1 Rabbit mAb (ab214034), EGF Receptor (D38B1) XP Rabbit mAb #4267 (Cell Signaling Technology, CST), p44/42 mitogen-activated protein kinase (Erk1/2) (137F5) rabbit mAb #4695 (CST), Phospho-p44/42 mitogen-activated protein kinase (Erk1/2) (Thr202/Tyr204) (D13.14.4E) XP Rabbit mAb #4370 (CST), beta-actin mouse mAb #60008-1-Ig (Proteintech), vinculin polyclonal antibody # 66305-1-Ig (E1E9V) (CST), Beclin1 mAb #66665-1-Ig (Proteintech), p70 S6 kinase antibody #9202 (CST), phospho-p70 S6 kinase (Thr389) Antibody #9205 (CST), and microtubule-associated protein 1 light chain 3 MAP1LC3 (LC3B) polyclonal antibody #14600-1-AP (Proteintech) were used for immunoblotting. shRNA and transfection Short hairpin RNA (shRNA) stably knocked down targeted genes were established by lentiviral infection in cell lines. .. HEK293T cells were transfected with the shRNA vector and packaging plasmid mix using PEI DNA transfection reagents (# PR40001, Proteintech).
Immunofluorescence:Article Title: Damage-sensing recruitment of a lipid phosphatase couples lysosomal membrane repair to proteostatic adaptation
Article Snippet: .. GAPDH (glyceraldehyde-3-phosphate dehydrogenase; mouse, Sigma-Aldrich, G8795, 1:4000); actin ( mouse, Sigma-Aldrich, Cat# A5441, 1:4000);Phospho-p70 S6 Kinase (Thr389) (rabbit, Cell signaling, Cat# 9205L,1:500), p70 S6 Kinase Antibody (rabbit, Cell Signaling, 9202L, 1:1000); PI4K2A (homemade); MTMR14 (homemade); dsRed/mScarlet (rabbit, Clontech, Cat# 632496, 1:1000); pULK(S757) (rabbit, Cell signaling, Cat# 6888, 1:1000); ULK1(rabbit, Cell signaling, Cat# 8054, 1:1000); p4E-BP1(S65) (rabbit, Cell signaling, Cat# 13443; 1:500); 4EBP1 (rabbit, Cell signaling, Cat# 9644; 1:500); pTFEB(S211) (rabbit, Cell signaling, Cat# 37681, 1:1000); TFEB (rabbit, Biomol, Cat# A303-673A, 1:1000); Anti-mouse HRP (Jackson, Cat# 115-035-003, 1:4000); Anti-rabbit HRP (Jackson, Cat# 111-035-003, 1:4000); Anti-mouse Alexa568 (IgM-specific) (Thermo, Cat# A21043, immunofluorescence 1:400); PtdIns(4)P (Echelon, Cat# z-p004, immunofluorescence 1:200); a-RXRXX[S/T] (Cell Signaling, Cat# 10001, 1:500); pS6 (Cell Signaling, Cat# 2215, 1:1000); pS6(S240/244) (Cell Signaling, Cat# 5364, 1:1000); puromycin (Kerafast, Cat# Kf-Ab02366-23.0, WB 1:500, immunofluorescence 1:2000); CHMP4B (Proteintech, 13683-1-AP, immunofluorescence 1:200); IST1 (proteintech, Cat# 19842-1-AP, immunofluorescence 1:200); Alix (Biolegend, Cat# 634501); PSMB5 (Thermo, Cat# PA1-977, immunofluorescence 1:200); Anti-mouse CF568 (Biotium, Cat# 20109-0.25ml, immunofluorescence 1:1000); Anti-rabbit Alexa 647 (Thermo, Cat# A31573, immunofluorescence 1:1000); Anti-rabbit Alexa 568 (Thermo/ Molecular Probes, Cat# A11036, immunofluorescence 1:1000); Anti-mouse Alexa 647 (life, Cat# A21238, immunofluorescence 1:1000); anti-Mouse IRDye 680 CW (LICOR, P/N 925-68070, 1:4000); anti-Rabbit IRDye 680 CW (LICOR, P/N 925-68180, 1:4000). .. L-Leucyl-L-Leucine methyl ester hydrobromide (LLOME, Sigma, Cat# L7393, 1mM, 2mM), Gly-Phe-β-naphthylamide (GPN, Cayman Chemical, Cat# 14634, 200μM), O-methyl-serine dodecylamide hydrochloride (MSDH, Avanti Polar Lipids, Cat# 850546, 50μM), Benzalkonium chloride (BAC, CAS# 63449-41-2, MP Biomedicals, Santa Ana, CA, USA, 5ug/mL), hydrogen peroxide (H 2 O 2 , Cat# ‘9681.2, Roth, 0.5mM), VPS34-IN1 (Selleckchem, Cat# S7980, 5μM), apilimod (Echelon Biosciences, Cat# B-0308, 50nM), rapamycin (Santa Cruz Biotechnology, Cat# sc-3504, 100nM), AZD8055 (AdooQ Bioscience/hölzel, Cat# A10114, 200nM), Anisomycin (Sigma, Cat# A9789, 30μM), LY2584702 (S6Ki, Selleckchem, Cat# S7704, 2μM, 5μM), MRT68921 dihydrochloride (MedChemExpress, Cat# 2080306-21-2, 1μM), bortezomib (MedChemExpress, Cat# 179324-69-7, 2μM), CCCP (Sigma, Cat# C2759-100MG, 10μM), Bafilomycin A1(Sigma, Cat# SML1661, 100nM or 200nM), nigericin (Invivogen, Cat# tlrl-nig,5μM), D-Mannitol (Sigma, Cat# M4125, 0.25M), Puromycin (InvivoGen, Cat# ant-pr-1, 2μM), Ionomycin (Calbiochem, Cat# 407952, 5μM), Doxycycline (Sigma, Cat# D-9891, 1μg/mL), Digitonin 5%(Thermo, Cat# BN20061, 20 mM).
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