Expressing:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Plasmid Preparation:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Cloning:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Mutagenesis:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Blocking Assay:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Binding Assay:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Construct:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Sequencing:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
FLAG-tag:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Introduce:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Generated:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-κB Activation
Article Snippet: .. The following transient expression plasmids were used: pCMV 126 3× flag -Cbl-b, -cIAP2, - ITCH, -NEDD4, and flag-βTrCP (27); pmCARMA1 ΔPRD 127 (49); pCMV flag- IKKβ-WT and pCMV flag-IKKβ-DA (Addgene plasmids 23298 and 128 11105) (25); pCR3 flag-BCL10 (a gift from M. Thome) (10); pCMV5B Flag-SMURF1 129 (Addgene plasmid 11752) (64); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247 , 130 Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) (19) pcDNA HA-131 TAK1 (created by ligating a double stranded oligonucleotide encoding an HA tag into the 132 5’ end of the multiple cloning site of pcDNA 3.1 (Invitrogen); site-directed mutagenesis 133 was then used (QuickChange II, Agilent) to generate restriction sites in pCMV-SPORT6 134 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA 135 backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 136 ligases, point mutations L122A, P123A, Y125A were introduced into TAK1 using site 137 directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-138 ΔTAB2/3 (to remove residues binding TAB2 and TAB3 (3), amino acids 509-533 of 139 TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-140 TAK1); pCMV SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3× 141 flag TAB1 (site directed mutagenesis was used to create restriction sites in pCMV 142 SPORT6 TAB1 that allowed direct cloning of TAB1 coding sequence downstream of an 143 N-terminal 3× flag tag in vector p3×FLAG-CMV-7.1 from Sigma-Aldrich); His-Ub was 144 a gift from K. Sabapathy (NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, 145 FHCRC); Igκ2-IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) (34); pRL-TK 146 (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of 147 human CARMA1 L225LI (a constitutively active mutant identified in a primary human 148 DLBCL) (20), site-directed mutagenesis was performed on pmCARMA1 to introduce 149 L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was 150 performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning 151 of CARMA1 coding sequence upstream of a C-terminal 3× flag epitope in plasmid 152 p3×FLAG-CMV-14 (Sigma-Aldrich); pIRES Puro Myc-BCL10 and pcDNA 153 destabilized-BCL10-6×Myc (d-BCL10) (generated by amplifying BCL10 coding 154 sequence from pCR3 flag-BCL10 using oligonucleotides with convenient restriction 155 sites. ..
Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
Clone Assay:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following antibodies were used: anti-BCL10 (331.3 and H-197), anti-phospho-extracellular signal-regulated kinase (anti-phospho-ERK) (Y204, E-4), anti-ERK (C-16), antiactin (C-2), antihemagglutinin (anti-HA) probe (Y-11), antiubiquitin (Fl-76), and anti-IκB-α (C-21) (Santa Cruz Biotechnology); anti-TAB1 (3225), anti-TAK1 (4505), anti-phospho-JNK (T183/Y185, 81E11), and anti-phospho-protein kinase D (anti-phospho-PKD) (S744/748, 2054) (Cell Signaling Technology); anti-myc epitope (9E10) and anti-Flag epitope (M2) (Sigma-Aldrich); pan anti-human/mouse cIAP1/2 (315301, R&D Systems); anti-ITCH (611198; BD Biosciences); anti-NEDD4 (07-049; Millipore); anti-chicken IgM (M4; Southern Biotech); anti-mouse IgM F(ab′) 2 (Jackson ImmunoResearch); IRDye800-conjugated anti-rabbit IgG (Rockland Immunochemicals); and Alexa Fluor 680-conjugated anti-mouse IgG (Invitrogen). .. The following transient expression plasmids were used: pCMV 3×Flag-Cbl-b, -cIAP2, - ITCH, -NEDD4, and Flag-βTrCP ( 25 ); pmCARMA1-ΔPRD ( 26 ); pCMV Flag-IKKβ-WT and pCMV Flag-IKKβ-DA (Addgene plasmids 23298 and 11105) ( 27 ); pCR3-Flag-BCL10 (a gift from M. Thome) ( 28 ); pCMV5B-Flag-SMURF1 (Addgene plasmid 11752) ( 29 ); pCMV-SPORT6 muTAK1 (IMAGE clone 3499247; Open Biosystems); pcDNA3-Flag-PKD2 (a gift from S. Sidorenko) ( 30 ); pcDNA HA-TAK1 (created by ligating a double-stranded oligonucleotide encoding an HA tag into the 5′ end of the multiple-cloning site of pcDNA 3.1 [Invitrogen]; site-directed mutagenesis was then used [QuikChange II; Agilent] to generate restriction sites in pCMV-SPORT6 TAK1 to allow in-frame cloning of TAK1 downstream of the HA tag in the pcDNA backbone); pcDNA HA-TAK1-AAYA (to block binding of TAK1 to TAB1 or HECT E3 ligases, point mutations L122A, P123A, and Y125A were introduced into TAK1 using site-directed mutagenesis of parental construct pcDNA HA-TAK1); pcDNA HA-TAK1-ΔTAB2/3 (to remove residues binding TAB2 and TAB3 [ 31 ], amino acids 509 to 533 of TAK1 were deleted by site-directed mutagenesis of parental construct pcDNA HA-TAK1); pCMV-SPORT6 hTAB1 (IMAGE clone 6202841; Open Biosystems); pCMV 3×Flag TAB1 (site-directed mutagenesis was used to create restriction sites in pCMV-SPORT6 TAB1 that allowed direct cloning of the TAB1 coding sequence downstream of an N-terminal 3×Flag tag in vector p3×Flag-CMV-7.1 [Sigma-Aldrich]); His-Ub (a gift from K. Sabapathy, NCC, Singapore); pCS 8× HA-Ub (a gift from J. Roberts, FHCRC); Igκ 2 -IFN-luciferase and pmCARMA1 (gifts from J. Pomerantz) ( 32 ); pRL-TK (Promega); pmCARMA1-L232LI (for expression of the murine CARMA1 version of human CARMA1 L225LI [a constitutively active mutant identified in a primary human DLBCL] [ 33 ]; site-directed mutagenesis was performed on pmCARMA1 to introduce the L232LI mutation); pCMV C3×F-CARMA1-ΔPRD (site-directed mutagenesis was performed on plasmid pmCARMA1 to create restriction sites allowing in-frame cloning of the CARMA1 coding sequence upstream of a C-terminal 3×Flag epitope in plasmid p3×Flag-CMV-14 [Sigma-Aldrich]); pIRES Puro myc-BCL10 and pcDNA destabilized BCL10-6×myc (d-BCL10) (generated by amplifying a BCL10 coding sequence from pCR3 Flag-BCL10 using oligonucleotides with convenient restriction sites; amplicons were digested and then cloned in-frame to N-terminal myc or C-terminal 6×myc epitope tags in the specified plasmid backbones, and sequence analysis showed cloning artifacts in pcDNA d-BCL10 6×myc [P3H, E11G mutations of BCL10, and 35 random amino acids after the 6×myc tag] that decreased the stability of wild-type pcDNA BCL10 6×myc). .. The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
other:Article Title: Kinase-Independent Feedback of the TAK1/TAB1 Complex on BCL10 Turnover and NF-?B Activation
Article Snippet: The following plasmids were use to generate lentiviral vectors (LVs) for stable expression of the genes of interest: pRRL MND-HA-TAK1 2A-GFP and pRRL MND HA-TAK1-AAYA 2A-GFP (coding sequences of pcDNA HA-TAK1 and pcDNA HA-TAK1-AAYA were isolated by restriction digest and cloned into pRRL MND 2A-GFP as described previously [ 34 ]); pRRL MND-HA-TAK1-K63W 2A-GFP; pRRL MND GFP ( 34 ); pRRL MND-myc-BCL10 2A-mCherry; pRRL MND-myc-BCL10-S138A 2A-mCherry; pRRL MND-mCherry; pGIPZ shTAB1 V3LHS_350795; 5′-TGCTGTTGACAGTGAGCGAAACGGCTATGATGGCAACCGATAGTGAAGCCACAGATGTATCGGTTGCCATCATAGCCGTTGTGCCTACTGCCTCGGA-3′; pGIPZ shTAK1 V2LHS_153758; 5′-TGCTGTTGACAGTGAGCGCGCAGATGAGCCATTACAGTATTAGTGAAGCCACAGATGTAATACTGTAATGGCTCATCTGCTTGCCTACTGCCTCGGA-3′ (Open Biosystems); and pGIPZ shScrambled (Open Biosystems).
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