Journal:

Article Title: CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16 INK4a -Independent Mechanisms ▿ †

doi: 10.1128/MCB.02286-06

Figure Lengend Snippet: Comparison of cyclin D1-CDK-CKI complexes in young and senescent HDFs. Equal amounts of cell lysate (2.5 mg) from young (Y), early senescence (ES), or late senescence (LS) TIG3 cells were subjected to gel filtration chromatography on a Superdex 200 column. Equivalent amounts (1/20th) of the individual fractions were separated by SDS-PAGE in a 12% acrylamide gel and immunoblotted with antisera against cyclin D1, CDK4, CDK6, p21CIP1, and p16INK4a. The lane on the left of each panel corresponds to a sample (12.5 μg) of total protein analyzed directly (i.e., without gel filtration).

Article Snippet: Rabbit polyclonal antibodies against CDK2 (sc-163), CDK4 (sc-601), CDK6 (sc-177), and p21 CIP1 (sc-397) were obtained from Santa Cruz.

Techniques: Comparison, Filtration, Chromatography, SDS Page, Acrylamide Gel Assay

Journal:

Article Title: CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16 INK4a -Independent Mechanisms ▿ †

doi: 10.1128/MCB.02286-06

Figure Lengend Snippet: Extension of HDF life span by wild-type and mutant versions of CDK4 and CDK6. (A) Hs68 cells were infected at PD40 with retroviruses encoding wild-type CDK4, the R24C mutant of CDK4, wild-type CDK6, the R31C mutant of CDK6, or the empty vector as indicated. Following selection in puromycin, the cell pools were analyzed by immunoblotting with antibodies against CDK4, CDK6, p16INK4a, and p21CIP1. MEK was used as a control for loading. Note that the samples were analyzed on the same gel, but the scanned images were subsequently edited for continuity of presentation. (B) Infected cell pools were passaged under standard conditions of tissue culture until they reached M1 senescence, as judged by failure to double in 4 weeks. The curves show cumulative PDs at each time point.

Article Snippet: Rabbit polyclonal antibodies against CDK2 (sc-163), CDK4 (sc-601), CDK6 (sc-177), and p21 CIP1 (sc-397) were obtained from Santa Cruz.

Techniques: Mutagenesis, Infection, Plasmid Preparation, Selection, Western Blot, Control

Journal:

Article Title: CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16 INK4a -Independent Mechanisms ▿ †

doi: 10.1128/MCB.02286-06

Figure Lengend Snippet: CDK4 and CDK6 can extend the life span of p16INK4a-deficient HDFs. (A) The Q34 strain of p16INK4a-deficient HDFs was infected at PD37 with retroviruses encoding wild-type CDK4, the R24C mutant of CDK4, wild-type CDK6, the R31C mutant of CDK6, or the empty vector, as indicated. Following selection, the infected cell pools were passaged under standard conditions of tissue culture until they reached M1 senescence. Curves show cumulative PDs at each time point. (B) Lysates prepared from infected cell pools were analyzed by immunoblotting with antibodies against CDK4, CDK6, p16INK4a, and p21CIP1. MEK was used as a control for loading. (C and D) The TIG3 strain of HDFs was infected at PD42 with retroviruses encoding CDK4 or Bmi1 or both, along with appropriate vector controls. Following selection, the infected cell pools were passaged under standard conditions of tissue culture until they reached M1 senescence. The curves show cumulative PDs at each time point. Lysates prepared from the infected cell pools were analyzed by immunoblotting with antibodies against CDK4, p16INK4a, and MEK.

Article Snippet: Rabbit polyclonal antibodies against CDK2 (sc-163), CDK4 (sc-601), CDK6 (sc-177), and p21 CIP1 (sc-397) were obtained from Santa Cruz.

Techniques: Infection, Mutagenesis, Plasmid Preparation, Selection, Western Blot, Control

Journal:

Article Title: CDK4 and CDK6 Delay Senescence by Kinase-Dependent and p16 INK4a -Independent Mechanisms ▿ †

doi: 10.1128/MCB.02286-06

Figure Lengend Snippet: Catalytically inactive versions of CDK4 and CDK6 are able to associate with cyclin D1, p21CIP1, and p16INK4a. TIG3 cells at PD42 were infected with retroviruses encoding HA-tagged versions of wild-type CDK4, CDK4D158N, wild-type CDK6, or CDK6D163N or empty-vector controls. (A) Following drug selection, cell lysates were prepared and samples (25 μg) of total protein were analyzed by SDS-PAGE in a 12% gel and immunoblotted with antibodies against CDK4 and CDK6. MEK served as a control for loading. As discussed in the text, ectopic expression of CDK4 resulted in multiple bands, the exact provenance of which remains unclear. Note also that the wild-type CDK4 construct contained two copies of the HA epitope. (B) Samples (500 μg) of protein were immunoprecipitated with a monoclonal antibody against the HA epitope, fractionated by SDS-PAGE, and immunoblotted for CDK4, CDK6, cyclin D1, p21CIP1, or p16INK4a as indicated.

Article Snippet: Rabbit polyclonal antibodies against CDK2 (sc-163), CDK4 (sc-601), CDK6 (sc-177), and p21 CIP1 (sc-397) were obtained from Santa Cruz.

Techniques: Infection, Plasmid Preparation, Selection, SDS Page, Control, Expressing, Construct, Immunoprecipitation

Journal: Engineering in Life Sciences

Article Title: Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

doi: 10.1002/elsc.201600033

Figure Lengend Snippet: Overview of different commercial miniaturized cultivation systems and their application

Article Snippet: Ninety‐six well approaches in standard MTPs were used for the rapamycin production in Streptomyces hygroscopius 50 , Glucansucrase from Leuconostoc mesenteroides mutants 51 , and recombinant human protein 1 from Chinese hamster ovary (CHO) cells 52 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 System Distributor Reactors Working volume (mL) PAT tools Reactor type Applications ambr Tap Biosystems up to 48 10–15 pH DOT STR mAbs from CHO cells 60 , 61 BioLector m2p‐labs GmbH 48 0.8–2.4 pH DOT MTP Glutathion‐S‐Transferase from E. coli 81 Organic acids from C. glutamicum 70 , 71 Recombinant parathyroid hormone (rPTH) from H. polymorpha 72 BioLevitator Hamilton Bonaduz AG 4 50 None Tube HeLa cells 174 bioREACTOR 48 2mag AG 48 8–15 pH DOT STR C. antarctica lipase B from Komagataella pastoris 64 Bioscreen C Pro Oy Growth Curves Ab Ltd 200 0.4 None MTP L. monocytogenes cell screening 175 S. cerevisiae cell screening 176 Cellstation Fluorometrix corp. 12 up to 35 pH DOT STR SP2/0 myeloma/mouse hybridoma cell cultures 177 DASbox Eppendorf AG 24 60–250 Sampler STR Cardiac differentiation of human pluripotent stem cells 178 HexaScreen Telstar Life Science Solutions 6 10–15 Sampler STR Suspended and adherent animal cell types 179 Micro‐24 Pall corp. 24 3–7 pH DOT MTP CHO cells 180 micro‐Flask Applikon Biotechnology up to 96 1 None MTP P. putida cultivations 181 micro‐Matrix Applikon Biotechnology 24 1–5 pH DOT MTP CD8 T‐cell line 182 Multifors INFORS HT 6 100–1000 Sampler STR α‐Ketoglutarate from Y. lipolytica 183 Hydrogen peroxide adapted bifidobacteria 184 SimCell Seahorse Bioscence Inc. 6 per array 0.7 pH DOT Microfluidic mAb from CHO cells 185 Xplorer HEL up to 8 1000–4000 Sampler STR E. coli fermentation 186 Open in a separate window Overview of different commercial miniaturized cultivation systems and their application Small‐scale stirred vessels 53 , 54 , 55 , 56 and bubble columns 57 , 58 bypass such losses in process information, whereas such systems rather represent a medium‐throughput approach.

Techniques: Recombinant

Journal: Engineering in Life Sciences

Article Title: Downstream process development strategies for effective bioprocesses: Trends, progress, and combinatorial approaches

doi: 10.1002/elsc.201600033

Figure Lengend Snippet: Overview of different commercial miniaturized systems in downstream process development and their applications

Article Snippet: Ninety‐six well approaches in standard MTPs were used for the rapamycin production in Streptomyces hygroscopius 50 , Glucansucrase from Leuconostoc mesenteroides mutants 51 , and recombinant human protein 1 from Chinese hamster ovary (CHO) cells 52 . table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 System Distributor Reactors Working volume (mL) PAT tools Reactor type Applications ambr Tap Biosystems up to 48 10–15 pH DOT STR mAbs from CHO cells 60 , 61 BioLector m2p‐labs GmbH 48 0.8–2.4 pH DOT MTP Glutathion‐S‐Transferase from E. coli 81 Organic acids from C. glutamicum 70 , 71 Recombinant parathyroid hormone (rPTH) from H. polymorpha 72 BioLevitator Hamilton Bonaduz AG 4 50 None Tube HeLa cells 174 bioREACTOR 48 2mag AG 48 8–15 pH DOT STR C. antarctica lipase B from Komagataella pastoris 64 Bioscreen C Pro Oy Growth Curves Ab Ltd 200 0.4 None MTP L. monocytogenes cell screening 175 S. cerevisiae cell screening 176 Cellstation Fluorometrix corp. 12 up to 35 pH DOT STR SP2/0 myeloma/mouse hybridoma cell cultures 177 DASbox Eppendorf AG 24 60–250 Sampler STR Cardiac differentiation of human pluripotent stem cells 178 HexaScreen Telstar Life Science Solutions 6 10–15 Sampler STR Suspended and adherent animal cell types 179 Micro‐24 Pall corp. 24 3–7 pH DOT MTP CHO cells 180 micro‐Flask Applikon Biotechnology up to 96 1 None MTP P. putida cultivations 181 micro‐Matrix Applikon Biotechnology 24 1–5 pH DOT MTP CD8 T‐cell line 182 Multifors INFORS HT 6 100–1000 Sampler STR α‐Ketoglutarate from Y. lipolytica 183 Hydrogen peroxide adapted bifidobacteria 184 SimCell Seahorse Bioscence Inc. 6 per array 0.7 pH DOT Microfluidic mAb from CHO cells 185 Xplorer HEL up to 8 1000–4000 Sampler STR E. coli fermentation 186 Open in a separate window Overview of different commercial miniaturized cultivation systems and their application Small‐scale stirred vessels 53 , 54 , 55 , 56 and bubble columns 57 , 58 bypass such losses in process information, whereas such systems rather represent a medium‐throughput approach.

Techniques: Clarification Assay, Solubility, Purification, Buffer Exchange, Molecular Weight, Diafiltration Assay, Plasmid Preparation, Biomarker Assay, Crystallization Assay, Binding Assay, Chromatography, DNA Extraction, Column Chromatography, Recombinant, Isolation