Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: Activation of both effector and initiator caspase is affected in PAK4 cell lines. HeLa control (pLPC) or PAK4 cells were left untreated (−) or stimulated with 10 ng of TNF-α per ml and 10 μg of CHX per ml (+), with TNF-α alone (T), or with CHX alone (C) for the indicated times in hours. Cells were harvested, and equal amounts of protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against caspase 8 (A and B), cleaved caspase 3 (C), or cFLIP (D). PAK4 expression levels and equal loading of the samples were detected by probing the extracts with hemagglutinin (HA) (E) and actin (F) antibodies. Specific bands of caspase 8 a and b proenzymes (proCaspase 8) and proteolytic products (p43/41), active p18 fragment, caspase 3 active p17 fragment, cFLIPshort, HA-PAK4, and actin are indicated by arrows.

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Activation Assay, SDS Page, Expressing

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: Cleavage of initiator caspase substrate Bid is affected in wild-type and kinase-dead PAK4 cell lines. HeLa control cells (pLPC) or cells expressing wild-type PAK4 (PAK4) or two independent kinase-dead cell lines (PAK4KMcl3 and PAK4KMclE2) were left unstimulated (−) or stimulated with 10 ng of TNF-α per ml and 10 μg of CHX per ml for the indicated times in hours (hs TNF/CHX). Cells were harvested, and equal amounts of protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against caspase 8 (A and B), cleaved caspase 3 (B), or Bid (D). PAK4 expression levels and equal loading of the samples were detected by probing the extracts with Myc (E), hemagglutinin (HA) (F), and actin (G) antibodies. Specific bands of caspase 8 a and b proenzymes (proCaspase 8) and proteolytic products (p43/41), active p18 fragment, caspase 3 active p17 fragment, or Bid full-length protein or p15 active fragment (cleaved Bid), wild-type HA-PAK4 or kinase-dead myc-PAK4, and actin are indicated by arrows. An unspecific reactive band (us) migrating near the p15 Bid fragment is indicated by an asterisk.

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Expressing, SDS Page

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: Stable cell lines expressing PAK4 are resistant to anti-FAS-induced apoptosis. HeLa control (pLPC) and wild-type (PAK4) or kinase-dead (PAK4KM) PAK4 cells were left unstimulated (ns) or treated with 500 ng of anti-Fas per ml and 10 μg of CHX per ml (F + C), for the indicated time in hours. Cells were harvested, and equal amounts of protein lysates were separated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with antibodies against PARP (A), caspase 8 (B), or Bid (C). PAK4 expression levels and equal loading of the samples were detected by probing the extracts with hemagglutinin (HA) and Myc antibodies (D) and actin antibodies (E). (Wild-type PAK4 is HA tagged, and PAK4KM is Myc tagged). Specific bands of PARP full-length p116 protein or cleaved p85 product, caspase 8 a and b proenzymes (proCaspase 8) and proteolytic products (p43/41), active p18 fragment, active Bid p15 fragment (cleaved Bid), wild type HA-PAK4 or kinase dead myc-PAK4 (PAK4), and actin are indicated by arrows. An unspecific reactive band (us) migrating near to the p15 Bid fragment is indicated by an asterisk.

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Stable Transfection, Expressing, SDS Page

Journal:

Article Title: Death Receptor-Induced Activation of Initiator Caspase 8 Is Antagonized by Serine/Threonine Kinase PAK4

doi: 10.1128/MCB.23.21.7838-7848.2003

Figure Lengend Snippet: PAK4-expressing cells show decreased Fas DISC formation. (A) The transfected TNFR/Fas chimeric receptor in highly expressed in PAK4 cell lines. HeLa control (pLPC) or wild-type PAK4 cells were transfected with empty vector (−) or the indicated amounts (in micrograms) of TNFR/Fas chimeric receptor (pCD120a/CD95) or GST (pEBG) expression vector. After 48 h, cells were harvested and equal volumes of protein lysates (1/100 of total extracts) (WCL) were analyzed by Western blotting as described below or used for immunopurification (Ip) with goat antibodies against the extracellular domain of p55 TNFR (αTNFR1) or preimmune goat serum (GS). Immunopurified complexes and whole-cell lysates were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with mouse antibodies against TNFR1 or GST. Specific bands are indicated by arrows on the right. Migration positions of the TNFR/Fas chimera are indicated by a brace. (B) Recruitment of caspase 8 is compromised in stable PAK4 cell lines. HeLa control (pLPC) or wild-type PAK4 cells were transfected with the indicated amounts (in micrograms) of TNFR/Fas chimeric receptor (DNA pCD120a/CD95) expression vector. After 48 h, cells were left untreated (−) or stimulated with 10 ng of TNF-α per ml for the indicated time in minutes and harvested. Equal volumes of protein lysates were analyzed by Western blotting as described below or used for immunopurification (1/100 of total extracts) with goat antibodies against the extracellular domain of p55 TNFR or preimmune goat serum. Immunopurified complexes were fractionated by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with mouse antibodies against caspase 8 or FADD. The blot probed with caspase 8 antibodies was stripped and reprobed with mouse antibodies against TNFR1 to verify TNFR1 and TNFR/Fas expression levels (as indicated at the left of the middle panel). Specific bands are indicated by arrows on the right. Migration positions of the TNFR/Fas chimera are indicated by a brace. Migration positions of IgG heavy and light chains are also indicated. To verify equal loading, the lysates (WCL) were analyzed by Western blotting for PAK4 (HA-PAK4), full-length caspase 8 (proCaspase8), FADD, and actin, as indicated. A diagram of the proteins interacting with the Fas intracellular domain of the TNFR/Fas chimeric receptor is also shown. (C) Quantitation of the caspase 8 signal in the DISC shows reduced caspase 8 protein per amount of receptor in PAK4 cells. The pro-caspase 8 bands of the top panel of panel B and the TNFR1 and TNFR/Fas bands (receptor) of the middle panel of panel B (pLPC and PAK4 lanes) were quantitated by densitometry (NIH Image). The densities of pixels in equal areas were measured, and backgrounds (measured in the GS Ip lanes) were subtracted. Receptor amounts for the single Ips were expressed as values relative to the amount of receptor present in the pLPC (ns) Ip lane. The relative amount of caspase 8 pixels shown in the graph was obtained by dividing the measured value of caspase 8 by its relative receptor levels (relative density of pixels). (The lowest relative value obtained [PAK4 ns lane] was similar to background levels and was set as the zero point of the y axis.)

Article Snippet: Monoclonal caspase 8 antibodies (clone 1C12), rabbit polyclonal cleaved caspase 3 (D175), and human Bid were from Cell Signaling; goat polyclonal TNFR1 antibodies were from R&D systems; monoclonal TNFR1 (H-5) and FLIPs/l (G11) antibodies were from Santa Cruz; monoclonal TRADD (clone 37), RIP (clone 38), and FADD (clone 1) were from BD Transduction Labs; monoclonal poly(ADP-ribose) polymerase (PARP) (clone 4C10-5) antibodies were from PharMingen; monoclonal CD95 Fas antibodies (clone CH-11) were from Immunotech; and monoclonal actin antibodies (clone AC-40) were from Sigma.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, Immu-Puri, SDS Page, Migration, Quantitation Assay

Journal: Leukemia & lymphoma

Article Title: Synergistic cytotoxicity of busulfan, melphalan, gemcitabine, panobinostat, and bortezomib in lymphoma cells

doi: 10.3109/10428194.2016.1157871

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

Article Snippet: The primary antibodies, their sources, dilutions, and antigen molecular weights are listed in (Supplementary Materials). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Antibodies Source (Catalogue #) Clone type Dilutions Molecular weight (kDa) AcH3K9 Active Motif/39137 pAb 3500 17 AKT Cell Signaling/4691 pAb 2000 60 ATM Santa Cruz/23921 mAb 800 250 BCL-2 DAKO/124 mAb 1500 26 c-MYC Cell Signaling/9402 pAb 2500 57–70 Cleaved Caspase 3 Cell Signaling/9661 pAb 2000 17,19 Caspase 8 Cell Signaling/9746 mAb 2000 43 Caspase 9 Cell Signaling/9502 pAb 2000 35–47 CHK2 Cell Signaling/2662 pAb 1000 62 HDAC4 Cell Signaling/5392 mAb 3000 140 KAP1 Bethyl Lab A300-275A pAb 2500 100–117 MCL-1 Santa Cruz/819 pAb 700 40 mTOR Cell Signaling/2983 pAb 2500 289 NOXA Calbiochem/0180 mAb 1500 15 p-ATM (Ser1981) Rockland/200–301-400 mAb 2000 250 p-AKT (Ser 473) Cell Signaling/4060 pAb 2500 60 p-CHK2 (Thr68) Cell Signaling/2661 pAb 2000 62 p-KAP1 (Ser824) Cell Signaling/4127 pAb 2500 100 p-mTOR (Ser2448) Cell Signaling/5536 pAb 1500 289 p-P53 (Ser 15) Cell Signaling/9284 pAb 2000 53 p-PI3K (Tyr199/Tyr458) Cell Signaling /4228 pAb 2500 60, 85 p-SMC1 (Ser957) Novus Biologicals/100–205 pAb 1500 140 P53 Santa Cruz/126 pAb 2000 53 PARP1 Santa Cruz /8007 mAb 1000 116 PI3K Santa Cruz/423 pAb 500 85 SMC1 Cell Signaling/4802 pAb 2500 140 XIAP Cell Signaling/2045 pAb 2000 53 α-Tubulin Cell Signaling/2144 pAb 1000 57 β-ACTIN Sigma/A5316 mAb 10000 42 γ-H2AX Millipore/2554898 mAb 3000 15 3MeH3K27 Active Motif/39155 pAb 3500 17 Open in a separate window List of primary antibodies, their sources, dilutions and molecular weights.

Techniques: Molecular Weight