calf intestinal alkaline phosphatase  (New England Biolabs)


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    Structured Review

    New England Biolabs calf intestinal alkaline phosphatase
    Calf Intestinal Alkaline Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calf intestinal alkaline phosphatase/product/New England Biolabs
    Average 99 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    calf intestinal alkaline phosphatase - by Bioz Stars, 2020-05
    99/100 stars

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    Related Articles

    Transfection:

    Article Title: Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis
    Article Snippet: .. Lysates of Vero cells that had been infected with wild-type HSV-2 186 at an MOI of 3 for 24 h and lysates of HEK293T cells that had been transfected with pEGFP-EF-1δ(F) were treated with calf intestinal alkaline phosphatase (CIP) (New England BioLabs) as described previously ( ). .. Vero and U2OS cells were infected with each of the recombinant viruses at an MOI of 0.0001, and plaque sizes were determined as described previously ( ).

    Amplification:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Ligation:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Infection:

    Article Title: Regulation of Herpes Simplex Virus 2 Protein Kinase UL13 by Phosphorylation and Its Role in Viral Pathogenesis
    Article Snippet: .. Lysates of Vero cells that had been infected with wild-type HSV-2 186 at an MOI of 3 for 24 h and lysates of HEK293T cells that had been transfected with pEGFP-EF-1δ(F) were treated with calf intestinal alkaline phosphatase (CIP) (New England BioLabs) as described previously ( ). .. Vero and U2OS cells were infected with each of the recombinant viruses at an MOI of 0.0001, and plaque sizes were determined as described previously ( ).

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Purification:

    Article Title: “Pocket-sized RNA-Seq”: A Method to Capture New Mature microRNA Produced from a Genomic Region of Interest
    Article Snippet: .. One microgram of bait RNA was therefore dephosphorylated using 1 U CIP (Alkaline Phosphatase, Calf Intestinal, New England Biolabs, Evry, France) at 37 °C for 60 min and RNAs were purified by phenol-chloroform extraction. .. RNA Sample Preparation RNA samples that will be hybridized to the bait were isolated from MCF-7 breast cancer cells or primary myoblasts with TRI reagent (Sigma) as previously described [ , ].

    Concentration Assay:

    Article Title: HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase *
    Article Snippet: .. Lysates (30 μg) were incubated with or without calf intestinal alkaline phosphatase at the concentration of 1 unit/μg of protein in CutSmart buffer, both obtained from New England Biolabs (Ipswich, MA). .. The reaction was supplemented with 1 m m protease inhibitor mixture and PMSF and incubated for 30 min at 37 °C.

    Incubation:

    Article Title: HIV-1-Tat Protein Inhibits SC35-mediated Tau Exon 10 Inclusion through Up-regulation of DYRK1A Kinase *
    Article Snippet: .. Lysates (30 μg) were incubated with or without calf intestinal alkaline phosphatase at the concentration of 1 unit/μg of protein in CutSmart buffer, both obtained from New England Biolabs (Ipswich, MA). .. The reaction was supplemented with 1 m m protease inhibitor mixture and PMSF and incubated for 30 min at 37 °C.

    Plasmid Preparation:

    Article Title: Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge
    Article Snippet: .. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, the plasmid Miniprep kit, and the DNA fragment gel extraction kit were purchased from New England Biolabs and used according to the manufacturer's specifications. .. B. abortus strain 2308 and the vaccine strain RB51 were obtained from the National Veterinary Services Laboratory, USDA (Ames, IA).

    De-Phosphorylation Assay:

    Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty
    Article Snippet: .. Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions. ..

    Article Title: Biochemical and Genetic Requirements for Function of the Immune Response Regulator BOTRYTIS-INDUCED KINASE1 in Plant Growth, Ethylene Signaling, and PAMP-Triggered Immunity in Arabidopsis [C] [C] [W]
    Article Snippet: .. Protein dephosphorylation was performed using calf intestinal alkaline phosphatase (CIP) according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units of CIP (as indicated)/μg protein. .. Total RNA was isolated with Trizol reagent according to the manufacturer’s instructions (Invitrogen).

    Polymerase Chain Reaction:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

    Gel Extraction:

    Article Title: Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge
    Article Snippet: .. Restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, the plasmid Miniprep kit, and the DNA fragment gel extraction kit were purchased from New England Biolabs and used according to the manufacturer's specifications. .. B. abortus strain 2308 and the vaccine strain RB51 were obtained from the National Veterinary Services Laboratory, USDA (Ames, IA).

    Derivative Assay:

    Article Title: Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor
    Article Snippet: .. Treatment of 10 μg of DNA from infected cells with 30 U of calf intestinal alkaline phosphatase at 37°C for 1 h (enzyme and reaction buffer from New England Biolabs [catalog no. 290S]) prior to Sau 3AI digestion, dilution, and ligation did not result in any significant reduction in the frequency of PCR products derived from amplification reactions on 250 pg of treated or untreated samples (19 and 6 products per 20 reactions, respectively). .. Sequencing of the products from both the treated and untreated samples showed no pattern of differences in the structures of the recombination joints.

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    New England Biolabs calf intestinal alkaline phosphatase cip
    Ser/Thr and Tyr autophosphorylation independently influence substrate specificity. Increasing amounts (8, 20, and 50 ng) of P-Clk/Sty (lanes 2 to 4), <t>PTP-Clk/Sty</t> (lanes 5 to 7), and <t>CIP-Clk/Sty</t> (lanes 8 to 10) were added to splicing reactions performed with S100 extracts in the presence of phosphatase inhibitors and ASF/SF2 (A) or SC35 (B). Reaction mixtures were deproteinized after 80 min, and RNAs were precipitated with ethanol. RNAs were fractionated by 5% denaturing PAGE and visualized by autoradiography.
    Calf Intestinal Alkaline Phosphatase Cip, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/calf intestinal alkaline phosphatase cip/product/New England Biolabs
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    calf intestinal alkaline phosphatase cip - by Bioz Stars, 2020-05
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    Ser/Thr and Tyr autophosphorylation independently influence substrate specificity. Increasing amounts (8, 20, and 50 ng) of P-Clk/Sty (lanes 2 to 4), PTP-Clk/Sty (lanes 5 to 7), and CIP-Clk/Sty (lanes 8 to 10) were added to splicing reactions performed with S100 extracts in the presence of phosphatase inhibitors and ASF/SF2 (A) or SC35 (B). Reaction mixtures were deproteinized after 80 min, and RNAs were precipitated with ethanol. RNAs were fractionated by 5% denaturing PAGE and visualized by autoradiography.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

    doi: 10.1128/MCB.23.12.4139-4149.2003

    Figure Lengend Snippet: Ser/Thr and Tyr autophosphorylation independently influence substrate specificity. Increasing amounts (8, 20, and 50 ng) of P-Clk/Sty (lanes 2 to 4), PTP-Clk/Sty (lanes 5 to 7), and CIP-Clk/Sty (lanes 8 to 10) were added to splicing reactions performed with S100 extracts in the presence of phosphatase inhibitors and ASF/SF2 (A) or SC35 (B). Reaction mixtures were deproteinized after 80 min, and RNAs were precipitated with ethanol. RNAs were fractionated by 5% denaturing PAGE and visualized by autoradiography.

    Article Snippet: Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions.

    Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography

    Autophosphorylation of Clk/Sty modulates kinase activity towards ASF/SF2 but not SRp40. (A) Increasing amounts of P-Clk/Sty (1, 5, and 20 ng) and equivalent amounts of PTP- and CIP-Clk/Sty were incubated with 1 μg of MBP under kinase conditions. Reactions were stopped by adding 1/2 volume of 3× SDS sample buffer, and proteins were fractionated by 10% SDS-PAGE and detected by autoradiography. (B and C) Increasing amounts of P-Clk/Sty (5, 20, and 100 ng) or equivalent amounts of PTP- and CIP-Clk/Sty were added to 1 μg of ASF/SF2 (B) or SRp40 (C) purified from E. coli and incubated for 30 min at 37°C. Reactions were processed as described for panel A except that proteins were transferred to nitrocellulose following PAGE. The extent of γ- 32 P incorporation was visualized by autoradiography (lanes 1 to 10). Subsequently, blots were incubated with the monoclonal antibody mAb104 (lanes 11 to 20) and reactivity was detected by chemiluminescence.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

    doi: 10.1128/MCB.23.12.4139-4149.2003

    Figure Lengend Snippet: Autophosphorylation of Clk/Sty modulates kinase activity towards ASF/SF2 but not SRp40. (A) Increasing amounts of P-Clk/Sty (1, 5, and 20 ng) and equivalent amounts of PTP- and CIP-Clk/Sty were incubated with 1 μg of MBP under kinase conditions. Reactions were stopped by adding 1/2 volume of 3× SDS sample buffer, and proteins were fractionated by 10% SDS-PAGE and detected by autoradiography. (B and C) Increasing amounts of P-Clk/Sty (5, 20, and 100 ng) or equivalent amounts of PTP- and CIP-Clk/Sty were added to 1 μg of ASF/SF2 (B) or SRp40 (C) purified from E. coli and incubated for 30 min at 37°C. Reactions were processed as described for panel A except that proteins were transferred to nitrocellulose following PAGE. The extent of γ- 32 P incorporation was visualized by autoradiography (lanes 1 to 10). Subsequently, blots were incubated with the monoclonal antibody mAb104 (lanes 11 to 20) and reactivity was detected by chemiluminescence.

    Article Snippet: Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions.

    Techniques: Activity Assay, Incubation, SDS Page, Autoradiography, Purification, Polyacrylamide Gel Electrophoresis

    Clk/Sty is autophosphorylated on Ser/Thr and Thr. (A) A total of 1 μg each of GST-Clk/Sty (lane 1) and a kinase-inactive mutant (GST-ClkR/Sty; lane 2) was fractionated by 10% SDS-PAGE and stained with Coomassie blue. The species indicated by the asterisk reflects an N-terminal truncation consisting of GST plus an approximately 10-kDa segment of Clk/Sty, which became phosphorylated in the wild-type but not the mutant sample. (B) A total of 0.5 μg of P-, PTP-, and CIP-Clk/Sty (lanes 1 to 3, respectively), was fractionated by 10% SDS-PAGE and stained with Coomassie blue. (C) A total of 20 to 50 ng of P-Clk/Sty (lane 1), PTP-Clk/Sty (lane 2), and CIP-Clk/Sty (lane 3) was fractionated by 10% SDS-PAGE and transferred to nitrocellulose. The blot was probed with antiphosphotyrosine antibody, and proteins were detected by chemiluminescence.

    Journal: Molecular and Cellular Biology

    Article Title: Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

    doi: 10.1128/MCB.23.12.4139-4149.2003

    Figure Lengend Snippet: Clk/Sty is autophosphorylated on Ser/Thr and Thr. (A) A total of 1 μg each of GST-Clk/Sty (lane 1) and a kinase-inactive mutant (GST-ClkR/Sty; lane 2) was fractionated by 10% SDS-PAGE and stained with Coomassie blue. The species indicated by the asterisk reflects an N-terminal truncation consisting of GST plus an approximately 10-kDa segment of Clk/Sty, which became phosphorylated in the wild-type but not the mutant sample. (B) A total of 0.5 μg of P-, PTP-, and CIP-Clk/Sty (lanes 1 to 3, respectively), was fractionated by 10% SDS-PAGE and stained with Coomassie blue. (C) A total of 20 to 50 ng of P-Clk/Sty (lane 1), PTP-Clk/Sty (lane 2), and CIP-Clk/Sty (lane 3) was fractionated by 10% SDS-PAGE and transferred to nitrocellulose. The blot was probed with antiphosphotyrosine antibody, and proteins were detected by chemiluminescence.

    Article Snippet: Tyr-dephosphorylated Clk/Sty (PTP-Clk/Sty) and unphosphorylated Clk/Sty (CIP-Clk/Sty) were prepared by adding 200 U of protein tyrosine phosphatase (PTP) (Boehringer Mannheim) and 40 U of calf intestinal alkaline phosphatase (CIP) (New England Biolabs), respectively, to 400 μg of P-Clk/Sty bound to glutathione beads, and dephosphorylation was carried out according to the manufacturers' instructions.

    Techniques: Mutagenesis, SDS Page, Staining

    BIK1 Conserved Residues and in Vitro and in Vivo Kinase Assays. (A) Structure of the BIK1 protein and comparison of residues in the ADs of BIK1 and related kinases. Gray region denotes the KD and black the AD. Residues noted in the BIK1 protein indicate those substituted for in planta assays. (B) Kinase activity of recombinant BIK1 and Ala substitution mutants produced in E. coli detected by autoradiogram. CCB, Coomassie blue staining. (C) and (D) BIK1 substitution mutants in vivo detected by a mobility shift on an HA-immunoblot or by phosphoserine/Thr-specific antibody. (E) BIK1 and MBP phosphorylation is abrogated by phosphatase treatment. Protein dephosphorylation was performed according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units CIP/μg protein (left) or (~2.5 μ/μg) (right). −, Buffer; +, CIP. In (C) and (D) , plants were treated with ACC (Ac) or flg22 (Fl) for 3 h and assayed for changes in BIK1 and MBP phosphorylation activity. In (C) to (E) , in vivo BIK1 phosphorylation was detected by ACC or flagellin-induced mobility shifts observable by HA-immunoblot. The top band corresponds to phosphorylated BIK1, which is migrating slower than the unphosphorylated form. MBP phosphorylation by BIK1 was detected by immunoblots with a phosphoserine/Thr-specific antibody.

    Journal: The Plant Cell

    Article Title: Biochemical and Genetic Requirements for Function of the Immune Response Regulator BOTRYTIS-INDUCED KINASE1 in Plant Growth, Ethylene Signaling, and PAMP-Triggered Immunity in Arabidopsis [C] [C] [W]

    doi: 10.1105/tpc.111.087122

    Figure Lengend Snippet: BIK1 Conserved Residues and in Vitro and in Vivo Kinase Assays. (A) Structure of the BIK1 protein and comparison of residues in the ADs of BIK1 and related kinases. Gray region denotes the KD and black the AD. Residues noted in the BIK1 protein indicate those substituted for in planta assays. (B) Kinase activity of recombinant BIK1 and Ala substitution mutants produced in E. coli detected by autoradiogram. CCB, Coomassie blue staining. (C) and (D) BIK1 substitution mutants in vivo detected by a mobility shift on an HA-immunoblot or by phosphoserine/Thr-specific antibody. (E) BIK1 and MBP phosphorylation is abrogated by phosphatase treatment. Protein dephosphorylation was performed according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units CIP/μg protein (left) or (~2.5 μ/μg) (right). −, Buffer; +, CIP. In (C) and (D) , plants were treated with ACC (Ac) or flg22 (Fl) for 3 h and assayed for changes in BIK1 and MBP phosphorylation activity. In (C) to (E) , in vivo BIK1 phosphorylation was detected by ACC or flagellin-induced mobility shifts observable by HA-immunoblot. The top band corresponds to phosphorylated BIK1, which is migrating slower than the unphosphorylated form. MBP phosphorylation by BIK1 was detected by immunoblots with a phosphoserine/Thr-specific antibody.

    Article Snippet: Protein dephosphorylation was performed using calf intestinal alkaline phosphatase (CIP) according to the manufacturer’s protocol (New England Biolabs) with ~1 to 2.5 units of CIP (as indicated)/μg protein.

    Techniques: In Vitro, In Vivo, Activity Assay, Recombinant, Produced, Staining, Mobility Shift, De-Phosphorylation Assay, Western Blot

    Both TagF and PppA domains repress T6SS activity independently of the PpkA-mediated TssL phosphorylation pathway in A. tumefaciens . A , Phos-tag SDS-PAGE analysis to detect the phosphorylation status of TssL-His. Shown is Western blot analysis of the same volumes of Ni-NTA resins (10 μl) associated with TssL-His from different strains treated with (+) or without (−) CIAP and examined by a specific antibody against His 6 . Total protein isolated from Δ tssL was a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His ( p-TssL-His ) and lower band indicating unphosphorylated TssL-His. B and C , Western blot analysis of the endogenous phosphorylation status of TssL (pTssL). Shown is Western blot analysis of total proteins isolated from WT C58, Δ ppkA , Δ tssL , or C58 harboring the vector pTrc200 (V) or various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The specific antibody for pTssL was generated against the 15-mer peptide ( 7 SSWQDLP pT VVEITEE 21 ), with phosphorylated Thr-14 of TssL underlined. RNA polymerase α subunit RpoA was an internal control. The proteins analyzed and molecular weight standards are on the left and right , respectively, and are indicated with an arrowhead when necessary. FL , full-length TagF-PppA proteins.

    Journal: The Journal of Biological Chemistry

    Article Title: TagF-mediated repression of bacterial type VI secretion systems involves a direct interaction with the cytoplasmic protein Fha

    doi: 10.1074/jbc.RA117.001618

    Figure Lengend Snippet: Both TagF and PppA domains repress T6SS activity independently of the PpkA-mediated TssL phosphorylation pathway in A. tumefaciens . A , Phos-tag SDS-PAGE analysis to detect the phosphorylation status of TssL-His. Shown is Western blot analysis of the same volumes of Ni-NTA resins (10 μl) associated with TssL-His from different strains treated with (+) or without (−) CIAP and examined by a specific antibody against His 6 . Total protein isolated from Δ tssL was a negative control. Phos-tag SDS-PAGE revealed the upper band indicating the phosphorylated TssL-His ( p-TssL-His ) and lower band indicating unphosphorylated TssL-His. B and C , Western blot analysis of the endogenous phosphorylation status of TssL (pTssL). Shown is Western blot analysis of total proteins isolated from WT C58, Δ ppkA , Δ tssL , or C58 harboring the vector pTrc200 (V) or various overexpressing plasmids grown in AB-MES (pH 5.5) liquid culture with specific antibodies. The specific antibody for pTssL was generated against the 15-mer peptide ( 7 SSWQDLP pT VVEITEE 21 ), with phosphorylated Thr-14 of TssL underlined. RNA polymerase α subunit RpoA was an internal control. The proteins analyzed and molecular weight standards are on the left and right , respectively, and are indicated with an arrowhead when necessary. FL , full-length TagF-PppA proteins.

    Article Snippet: Dephosphorylation and Phos-tag SDS-PAGE analyses Dephosphorylation analysis by calf intestinal alkaline phosphatase (CIAP) was performed according to the user manual (New England Biolabs, Beverly, MA) with minor modifications as described previously ( ).

    Techniques: Activity Assay, SDS Page, Western Blot, Isolation, Negative Control, Plasmid Preparation, Generated, Molecular Weight

    Western blot analysis of Panx2 in tissue and post-translational modifications in tissue culture cells. (A) Incubation of the Panx2 antibody with the immunizing peptide prior to western blotting eliminated the labeling of untagged Panx2-WT proteins stably expressed in HeLa cells. Western blots for alpha-tubulin levels shown below the Panx2 immunoblots served as loading controls for these lysates. (B) The expected mass of Panx2 monomers based on their amino acid sequence is ~74 kDa and western blot analysis of HeLa cell lysates stably expressing untagged rat Panx2 showed a major band at about this molecular mass (left and right hand lanes). Some lower bands were also observed at about 45 kDa. In tissue lysates from rat and mouse brains, a weaker ~74 kDa band was observed as well as few faster migrating bands that may represent caspase cleaved Panx2 species. (C) Cell lysates from three cell lines, MDCK, HEK 293T, and HeLa, each stably expressing HA-tagged Panx2, were treated with either Z-VAD-OMe-FMK (Z-VAD) to inhibit caspase-dependent cleavage, PNGase to reduce glycosylation, or CIAP for protein dephosphorylation. We did not see any significant shift in the bands indicating that at least in these cell types, HA-tagged Panx2 is not highly phosphorylated or glycosylated. (D) However, cell lysates from HeLa, and MDCK, each stably expressing untagged Panx2-WT revealed a slight shift in banding pattern following PNGase treatment. As positive control, lysates from MDCK cells stably expressing untagged Panx1-WT showed significant band shifts after PNGase treatment. Western blots shown here are representative of at least three independent experiments per group.

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane

    doi: 10.3389/fncel.2014.00468

    Figure Lengend Snippet: Western blot analysis of Panx2 in tissue and post-translational modifications in tissue culture cells. (A) Incubation of the Panx2 antibody with the immunizing peptide prior to western blotting eliminated the labeling of untagged Panx2-WT proteins stably expressed in HeLa cells. Western blots for alpha-tubulin levels shown below the Panx2 immunoblots served as loading controls for these lysates. (B) The expected mass of Panx2 monomers based on their amino acid sequence is ~74 kDa and western blot analysis of HeLa cell lysates stably expressing untagged rat Panx2 showed a major band at about this molecular mass (left and right hand lanes). Some lower bands were also observed at about 45 kDa. In tissue lysates from rat and mouse brains, a weaker ~74 kDa band was observed as well as few faster migrating bands that may represent caspase cleaved Panx2 species. (C) Cell lysates from three cell lines, MDCK, HEK 293T, and HeLa, each stably expressing HA-tagged Panx2, were treated with either Z-VAD-OMe-FMK (Z-VAD) to inhibit caspase-dependent cleavage, PNGase to reduce glycosylation, or CIAP for protein dephosphorylation. We did not see any significant shift in the bands indicating that at least in these cell types, HA-tagged Panx2 is not highly phosphorylated or glycosylated. (D) However, cell lysates from HeLa, and MDCK, each stably expressing untagged Panx2-WT revealed a slight shift in banding pattern following PNGase treatment. As positive control, lysates from MDCK cells stably expressing untagged Panx1-WT showed significant band shifts after PNGase treatment. Western blots shown here are representative of at least three independent experiments per group.

    Article Snippet: Analysis of post-translational modifications of Panx1 and Panx2 expressing tissue culture cells Whole cell lysates from HEK 293T, HeLa and MDCK cells stably expressing Panx2-HA, Panx2-WT, or Panx1-WT proteins were incubated with 10 units of calf intestinal alkaline phosphatase (CIAP) for 5 h at 37°C; or with 1500 units of N -glycosidase F (PNGase F) (New England Biolabs, Beverly, MA) for 5 h at 37°C at pH 7.5 following procedures we used in Boassa et al. ( ) or 50 μM pan-caspase inhibitor Z-VAD-OMe-FMK for 5 h at 37°C (Millipore, Billerica, MA) following a procedures described in Penuela et al. ( ).

    Techniques: Western Blot, Incubation, Labeling, Stable Transfection, Sequencing, Expressing, De-Phosphorylation Assay, Positive Control