rnase assay buffer (SignalChem)

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SignalChem rnase assay buffer

Fortilin interacts with the cytosolic domain of IRE1α and inhibits its protein kinase and <t>RNase</t> activities. a Proximity ligation assay (PLA) shows a specific interaction between fortilin and P-IRE1α in EGF-SubA-treated PC3 cells. The cells were treated with 2 nM EGF-SubA for 24 h and subjected to PLA, using anti-IREα and anti-P-IRE1α antibodies to evaluate fortilin-IRE1α and fortilin-P-IRE1α interaction, respectively. PLA interaction indices were calculated by dividing the number of red dots by the number of nuclei, expressed as means ± s.d. ( n = 3), and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. Scale bar = 10 µm. b Fortilin co-immunoprecipitates P-IRE1α. PC3 cells were treated with 2 nM EGF-SubA for 24 h, lysed and subjected to immunoprecipitation (IP). c Domain structure of human IRE1α. Human IRE1α consists of the ER luminal domain (aa 1–443), transmembrane domain (aa 444–464), linker region (aa 465–567), kinase domain (aa 568–833), and endoribonuclease (RNase) domain (aa 836–997). The <t>following</t> <t>recombinant</t> proteins were used for biolayer interferometry: full-length IRE1α (aa 1-977), IRE1α-Myc-DDK (aa 1–977); IRE1α-L, GST-IRE1α (aa 1–70); IRE1α-TM, GST-IRE1α (aa 401–500); and IRE1α-C, GST-IRE1α (aa 468–977). d – h Fortilin binds to P-IRE1α through its cytosolic domain. Biotinylated fortilin was immobilized to the streptavidin biosensor. Recombinant IRE1α, either full-length or fragment, was applied to the biosensor at various concentrations, and dissociation constants (Kds, expressed as mean ± s.d., n = 3) were derived. i Lowest energy binding pose of fortilin ( blue ) with cytosolic domain of IRE1α ( green ) (the right panel ) presented with that of a fortilin-fortilin dimer (the left panel ). j Intermolecular interactions between phosphorylated serine724 (pS 724 ) and serine726 (pS 726 ) of the cytosolic domain of IRE1α with lysine residues (K 19 and K 34 ) of fortilin. k Fortilin inhibits the RNase activity of IRE1α. An in vitro IRE1α RNase activity assay was performed by incubating IRE1α with human recombinant fortilin and the substrate fluorescently tagged XBP1 RNA stem loop, the cleavage of which would allow the fluorescein amidite (FAM) to fluoresce. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. l Fortilin inhibits the kinase activity of IRE1α. An in vitro IRE1α kinase activity assay was performed by incubating IRE1α with [γ- 33 P]ATP, recombinant fortilin, and myelin basic protein (MBP) as a substrate of the kinase in the kinase reaction buffer. The phosphorylation index was calculated by dividing the radioactivity of MBP for a given fortilin concentration by that of the vehicle control and expressed as means ± s.d. ( n = 2) from which half maximal inhibitory concentration (IC 50 ) was calculated

Rnase Assay Buffer, supplied by SignalChem, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pla+blocking+solution/Kinase+Assay+Buffer+II/pmc05446404-404-19-6
Average 92 stars, based on 28 article reviews

rnase assay buffer - by Bioz Stars, 2026-09

92/100 stars

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1) Product Images from "Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death"

Article Title: Fortilin binds IRE1α and prevents ER stress from signaling apoptotic cell death

Journal: Nature Communications

doi: 10.1038/s41467-017-00029-1

Fortilin interacts with the cytosolic domain of IRE1α and inhibits its protein kinase and RNase activities. a Proximity ligation assay (PLA) shows a specific interaction between fortilin and P-IRE1α in EGF-SubA-treated PC3 cells. The cells were treated with 2 nM EGF-SubA for 24 h and subjected to PLA, using anti-IREα and anti-P-IRE1α antibodies to evaluate fortilin-IRE1α and fortilin-P-IRE1α interaction, respectively. PLA interaction indices were calculated by dividing the number of red dots by the number of nuclei, expressed as means ± s.d. ( n = 3), and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. Scale bar = 10 µm. b Fortilin co-immunoprecipitates P-IRE1α. PC3 cells were treated with 2 nM EGF-SubA for 24 h, lysed and subjected to immunoprecipitation (IP). c Domain structure of human IRE1α. Human IRE1α consists of the ER luminal domain (aa 1–443), transmembrane domain (aa 444–464), linker region (aa 465–567), kinase domain (aa 568–833), and endoribonuclease (RNase) domain (aa 836–997). The following recombinant proteins were used for biolayer interferometry: full-length IRE1α (aa 1-977), IRE1α-Myc-DDK (aa 1–977); IRE1α-L, GST-IRE1α (aa 1–70); IRE1α-TM, GST-IRE1α (aa 401–500); and IRE1α-C, GST-IRE1α (aa 468–977). d – h Fortilin binds to P-IRE1α through its cytosolic domain. Biotinylated fortilin was immobilized to the streptavidin biosensor. Recombinant IRE1α, either full-length or fragment, was applied to the biosensor at various concentrations, and dissociation constants (Kds, expressed as mean ± s.d., n = 3) were derived. i Lowest energy binding pose of fortilin ( blue ) with cytosolic domain of IRE1α ( green ) (the right panel ) presented with that of a fortilin-fortilin dimer (the left panel ). j Intermolecular interactions between phosphorylated serine724 (pS 724 ) and serine726 (pS 726 ) of the cytosolic domain of IRE1α with lysine residues (K 19 and K 34 ) of fortilin. k Fortilin inhibits the RNase activity of IRE1α. An in vitro IRE1α RNase activity assay was performed by incubating IRE1α with human recombinant fortilin and the substrate fluorescently tagged XBP1 RNA stem loop, the cleavage of which would allow the fluorescein amidite (FAM) to fluoresce. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. l Fortilin inhibits the kinase activity of IRE1α. An in vitro IRE1α kinase activity assay was performed by incubating IRE1α with [γ- 33 P]ATP, recombinant fortilin, and myelin basic protein (MBP) as a substrate of the kinase in the kinase reaction buffer. The phosphorylation index was calculated by dividing the radioactivity of MBP for a given fortilin concentration by that of the vehicle control and expressed as means ± s.d. ( n = 2) from which half maximal inhibitory concentration (IC 50 ) was calculated

Figure Legend Snippet: Fortilin interacts with the cytosolic domain of IRE1α and inhibits its protein kinase and RNase activities. a Proximity ligation assay (PLA) shows a specific interaction between fortilin and P-IRE1α in EGF-SubA-treated PC3 cells. The cells were treated with 2 nM EGF-SubA for 24 h and subjected to PLA, using anti-IREα and anti-P-IRE1α antibodies to evaluate fortilin-IRE1α and fortilin-P-IRE1α interaction, respectively. PLA interaction indices were calculated by dividing the number of red dots by the number of nuclei, expressed as means ± s.d. ( n = 3), and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. Scale bar = 10 µm. b Fortilin co-immunoprecipitates P-IRE1α. PC3 cells were treated with 2 nM EGF-SubA for 24 h, lysed and subjected to immunoprecipitation (IP). c Domain structure of human IRE1α. Human IRE1α consists of the ER luminal domain (aa 1–443), transmembrane domain (aa 444–464), linker region (aa 465–567), kinase domain (aa 568–833), and endoribonuclease (RNase) domain (aa 836–997). The following recombinant proteins were used for biolayer interferometry: full-length IRE1α (aa 1-977), IRE1α-Myc-DDK (aa 1–977); IRE1α-L, GST-IRE1α (aa 1–70); IRE1α-TM, GST-IRE1α (aa 401–500); and IRE1α-C, GST-IRE1α (aa 468–977). d – h Fortilin binds to P-IRE1α through its cytosolic domain. Biotinylated fortilin was immobilized to the streptavidin biosensor. Recombinant IRE1α, either full-length or fragment, was applied to the biosensor at various concentrations, and dissociation constants (Kds, expressed as mean ± s.d., n = 3) were derived. i Lowest energy binding pose of fortilin ( blue ) with cytosolic domain of IRE1α ( green ) (the right panel ) presented with that of a fortilin-fortilin dimer (the left panel ). j Intermolecular interactions between phosphorylated serine724 (pS 724 ) and serine726 (pS 726 ) of the cytosolic domain of IRE1α with lysine residues (K 19 and K 34 ) of fortilin. k Fortilin inhibits the RNase activity of IRE1α. An in vitro IRE1α RNase activity assay was performed by incubating IRE1α with human recombinant fortilin and the substrate fluorescently tagged XBP1 RNA stem loop, the cleavage of which would allow the fluorescein amidite (FAM) to fluoresce. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. l Fortilin inhibits the kinase activity of IRE1α. An in vitro IRE1α kinase activity assay was performed by incubating IRE1α with [γ- 33 P]ATP, recombinant fortilin, and myelin basic protein (MBP) as a substrate of the kinase in the kinase reaction buffer. The phosphorylation index was calculated by dividing the radioactivity of MBP for a given fortilin concentration by that of the vehicle control and expressed as means ± s.d. ( n = 2) from which half maximal inhibitory concentration (IC 50 ) was calculated

Techniques Used: Proximity Ligation Assay, Two Tailed Test, Immunoprecipitation, Recombinant, Derivative Assay, Binding Assay, Activity Assay, In Vitro, Kinase Assay, Radioactivity, Concentration Assay

Protection by fortilin against EGF-SubA-induced liver damage is mediated by its ability to block the activation of the IRE1α-JNK apoptosis pathway. a Experimental protocol. Five-week-old male fortilin WT-liver or fortilin KO-liver mice ( n = 6) were pretreated by vehicle or the IRE1α kinase inhibiting RNAse attenuator-6 (KIRA6) for 3 days, challenged by EGF-SubA once on the third day, and treated by vehicle or KIRA6 for an additional 6 days. b KIRA6 protects both fortilin WT-liver and fortilin KO-liver mice against EGF-SubA-induced liver damage. The sera from EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either vehicle or KIRA6, were assayed for ALT. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. c The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit similar gross appearance when treated with KIRA6. Scale bar = 10 mm. d Lack of change in expression and phosphorylation patterns of the PERK and ATF6 pathway proteins in the EGF-SubA-challenged livers, regardless of the status of fortilin or of KIRA6 treatment. The total lysates from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either KIRA6 or vehicle, were subjected to quantitative IB using the indicated antibodies. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. e The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of IRE1α and JNK phosphorylation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. * P < 0.05; *** P < 0.005. f The livers of EGF-SubA-challenged, KIRA6-treated, fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of XBP1 splicing. The total RNA from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice were assayed for the amounts of XBP1s and XBP1u . Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. g The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit equal amounts of apoptosis when treated with KIRA6. Paraffin sections from the livers of EGF-SubA-treated fortilin WT-liver and fortilin KO-liver mice ( n = 6 each) for cleaved lamin. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. ** P < 0.01; F.C . fold change. Scale bar = 50 µm. h , i The immunostaining of the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit the same degree of IRE1α pathway activation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. *** P < 0.005. Scale bar = 50 µm. j Proposed model of the role of fortilin in ER stress-induced apoptosis

Figure Legend Snippet: Protection by fortilin against EGF-SubA-induced liver damage is mediated by its ability to block the activation of the IRE1α-JNK apoptosis pathway. a Experimental protocol. Five-week-old male fortilin WT-liver or fortilin KO-liver mice ( n = 6) were pretreated by vehicle or the IRE1α kinase inhibiting RNAse attenuator-6 (KIRA6) for 3 days, challenged by EGF-SubA once on the third day, and treated by vehicle or KIRA6 for an additional 6 days. b KIRA6 protects both fortilin WT-liver and fortilin KO-liver mice against EGF-SubA-induced liver damage. The sera from EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either vehicle or KIRA6, were assayed for ALT. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. NS not statistically significant; *** P < 0.005. c The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit similar gross appearance when treated with KIRA6. Scale bar = 10 mm. d Lack of change in expression and phosphorylation patterns of the PERK and ATF6 pathway proteins in the EGF-SubA-challenged livers, regardless of the status of fortilin or of KIRA6 treatment. The total lysates from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice, treated with either KIRA6 or vehicle, were subjected to quantitative IB using the indicated antibodies. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. e The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of IRE1α and JNK phosphorylation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. * P < 0.05; *** P < 0.005. f The livers of EGF-SubA-challenged, KIRA6-treated, fortilin WT-liver and fortilin KO-liver mice exhibit a similar degree of XBP1 splicing. The total RNA from the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice were assayed for the amounts of XBP1s and XBP1u . Data were expressed as means ± s.d. ( n = 4) and analyzed by two-tailed unpaired t -test. *** P < 0.005. g The livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit equal amounts of apoptosis when treated with KIRA6. Paraffin sections from the livers of EGF-SubA-treated fortilin WT-liver and fortilin KO-liver mice ( n = 6 each) for cleaved lamin. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. ** P < 0.01; F.C . fold change. Scale bar = 50 µm. h , i The immunostaining of the livers of EGF-SubA-challenged fortilin WT-liver and fortilin KO-liver mice exhibit the same degree of IRE1α pathway activation when treated with KIRA6. Data were expressed as means ± s.d. ( n = 6) and analyzed by two-tailed unpaired t -test. *** P < 0.005. Scale bar = 50 µm. j Proposed model of the role of fortilin in ER stress-induced apoptosis

Techniques Used: Blocking Assay, Activation Assay, Two Tailed Test, Expressing, Immunostaining

other:

Article Title: Corynoline inhibits esophageal squamous cell carcinoma growth via targeting Pim-3.
Article Snippet: Kinase buffer was purchased from SignalChem (British Columbia, Canada, Cat# K02–09).

Incubation:

Article Title: PKA-Independent Vasopressin Signaling in Renal Collecting Duct
Article Snippet: .. All peptides (custom peptides, 20 nmol; standard peptides, 10 nmole) were incubated with various purified enzymes individually (i.e. AMPK, SIK2) in kinase reaction buffer (25 mM MOPS, 25 mM MgCl 2 , 2 mM EDTA, 12.5 mM β-glycerol-phosphate) and supplemented with 100 μM ATP and 0.25 mM DTT for 1 h at 30 °C (all components were from Signal Chem, Richmond, BC, Canada). .. The proteins were transferred to nitrocellulose membranes and probed with primary antibodies from Cell Signaling Technology (Danvers, MA) at indicated dilutions: anti-AMPKα (1:1000, isotype: Rabbit, Cat. No. 5832), anti-phospho AMPKα (Thr172) (1: 1000, isotype: Rabbit IgG, Cat. No. 50081).

Purification:

Lysis:

Article Title: Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer
Article Snippet: .. After washes in lysis buffer, beads were resuspended in kinase reaction buffer (25 mM MOPS, pH 7.2, 12.5 mM β-glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA, and 0.25 mM DTT) and the kinase (Akt1/2/3 or SGK1, SignalChem) and ATP were added at final concentrations of 50nM and 200 μM respectively. ..

Article Title: Methylation of the chromatin modifier KMT2D by SMYD2 contributes to therapeutic response in hormone-dependent breast cancer.
Article Snippet: .. After washes in lysis buffer, beads were resuspended in kinase reaction buffer (25 mM MOPS, pH 7.2, 12.5 mM b-glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA, and 0.25 mM DTT) and the kinase (Akt1/2/3 or SGK1, SignalChem) and ATP were added at final concentrations of 50nM and 200 mM respectively. ..

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