Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: Kinase dead RIPK1 D138N mice are not protected from APAP toxicity in vivo while RIPK1 knockdown in RIPK1 D138N mice is protective . A and B , WT and RIPK1 D138N mice were fasted overnight and treated with APAP (300 mg/kg) and euthanized at 24 h. A , serum ALT (U/L). B , representative histology H&E (4x). C and D , RIPK1 D138N mice were treated with RIPK1 or Control ASO five times, subsequently injected with APAP (300 mg/kg) after overnight fast and euthanized at 24 h. C , serum ALT (U/L). D , representative histology H&E (4x). E , WB of RIPK1 and loading control in RIPK1 D138N mice treated with RIPK1 or Control ASO. ∗ p value ≤ 0.05 RIPK1 versus Control ASO treated mice (N = 11–12/group). Results of at least three independent experiments. ALT, alanine aminotransferase; ASO, antisense oligonucleotide; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RIPK1, receptor interacting protein kinase 1; RIPK1 D138N , RIPK1 kinase dead knock-in mice; WT, wild type.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: In Vivo, Knockdown, Control, Injection, Knock-In

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: Hepatocyte-specific RIPK1 knockout (RIPK1 HepCKO ) protects against APAP toxicity in vivo . RIPK1 flx/flx mice were treated with AAV8-TBG-iCRE or AAV8-TBG-eGFP for hepatocyte-specific knockout (RIPK1 HepCKO ). Ten days later mice were treated with PBS or APAP (300 mg/kg) after overnight fast and euthanized at 1 h, 3 h, (for GSH and NAPQI adducts) or 24 h (for ALT). A , serum ALT U/L at 24 h (N = 12–13/group). B , representative histology H&E (4x). C , WB of RIPK1 and loading control showing protein knockout. D , representative WB of NAPQI protein adducts, densitometry, and loading control, 1 h and 3 h (N = 6/group). E , GSH at 1 h and 3 h using a colorimetric recycling assay expressed as μmoles/gram liver (N = 3/group). ∗ p value ≤0.05 RIPK1 HepCKO versus RIPK1-flx/flx mice ∗∗ p value ≤0.01 RIPK1 HepCKO versus RIPK1-flx/flx. Results of at least three independent experiments. ALT, alanine aminotransferase; APAP, acetaminophen; Flx, Floxed; GFP, green fluorescent protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NAPQI, N-acetyl-p-benzoquinone imine; N.S., not significant; PBS, phosphate buffered saline; RIPK1, receptor interacting protein kinase 1.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Knock-Out, In Vivo, Control, Saline

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: RIPK1 HepCKO results in dampening of JNK activation, less p-JNK translocation to the mitochondria, and decreased pMKK4 . RIPK1 flx/flx or RIPK1 HepCKO mice were fasted overnight and treated with APAP 300 mg/kg and euthanized 1 h or 3 h later. A , WB of liver lysates for pJNK, JNK, pMKK4, MKK4, ASK1, and GAPDH as loading control. B , densitometry for pJNK and pMKK4 normalized to their corresponding total protein expression and ASK1 normalized to loading control. C , WB of mitochondrial fraction for pJNK and loading control. D , densitometry of mitochondrial pJNK normalized to loading control. ∗ p value ≤0.05 RIPK1 HepCKO versus RIPK1 flx/flx mice and ∗∗ p value ≤0.01 RIPK1 HepCKO versus RIPK1 flx/flx mice. Flx, Floxed; JNK, c Jun N-terminal kinase; MKK4, mitogen-activated protein kinase 4; ASK1, apoptosis signal regulating kinase 1; RIPK1, receptor interacting protein kinase 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PHB1, prohibitin-1; PBS, phosphate buffered saline; APAP, acetaminophen. (N = 4/group). Results of at least three independent experiments.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Activation Assay, Translocation Assay, Control, Expressing, Saline

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: RIPK1 knockdown and knockout increases A20 protein expression . WT mice were treated with RIPK1 or Control ASO (50 mg/kg) five times, subsequently injected with APAP (300 mg/kg) after fasting and euthanized at 1 h, 2 h, or 4 h. A , representative WB of A20, RIPK1 and loading control. B , RIPK1 flx/flx or RIPK1 HepCKO mice were treated with PBS or APAP (300 mg/kg) after overnight fast and euthanized at 1 h and 3 h. Representative WB of A20, RIPK1 and loading control. C , relative A20 transcript levels normalized to TBP and RIPK1 flx/flx PBS (N = 5/group). ∗∗ p value ≤0.01 RIPK1 HepCKO versus RIPK1 flx/flx . Results of at least three independent experiments. APAP, acetaminophen; ASO, antisense oligonucleotide; CTRL, control; NS, not significant; RIPK1, receptor interacting protein kinase 1; TBP, TATA box binding protein.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Knockdown, Knock-Out, Expressing, Control, Injection, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: Mice with hepatocyte-specific knockout of A20 (A20 HepCKO ) display worse liver injury following APAP compared with littermate controls (A20 flx/flx ) . A20 flx/flx mice were treated with AAV8-TBG-iCRE or AAV8-TBG-eGFP for hepatocyte-specific knockout (A20 HepCKO ), after 7 days mice were fasted overnight and treated with APAP 300 mg/kg and euthanized 3 h or 24 h later. A , serum ALT U/L at 24 h (N = 10–11/group). B , representative Histology H&E (4x). C , representative WB of liver lysates for A20 and loading control. D , representative WB of RIPK1 and loading control. E , representative WB of NAPQI protein adducts, densitometry, and loading control (N = 4/group). F , GSH using a colorimetric recycling assay expressed as μmoles/gram liver. (N = 3/group). ∗ p value ≤0.05 A20 HepCKO versus A20-flx/flx mice. Results of at least three independent experiments. ALT, alanine aminotransferase; APAP, acetaminophen; Flx, floxed; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NAPQI, N-acetyl-p-benzoquinone imine; N.S., not significant; PBS, phosphate buffered saline; RIPK1, receptor interacting protein kinase-1.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Knock-Out, Control, Saline

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: A20 HepCKO results in sustained JNK activation through increased ASK1 protein . A20 flx/flx or A20 HepCKO mice were fasted overnight and treated with APAP 300 mg/kg and euthanized 3 h later. A , WB of liver lysates for p JNK, JNK, pMKK4, MKK, ASK1, and GAPDH as loading control. B , densitometry for pJNK and pMKK4 normalized to their total protein expression and ASK1 normalized to loading control. C , WB of mitochondrial fraction for pJNK and PHB1, loading control. D , densitometry is shown as mitochondrial pJNK normalized to PHB1 loading control. E , relative transcript levels of ASK1 normalized to TBP and RIPK1 flx/flx PBS (N = 5/group). ∗ p value ≤0.05 A20 HepCKO versus A20-flx/flx mice; ∗∗ p value ≤0.01 A20 HepCKO versus A20-flx/flx mice. (N = 4/group). Results of at least three independent experiments. APAP, acetaminophen; ASK1, apoptosis signal regulating kinase 1; Flx, floxed; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; JNK, c Jun N-terminal kinase; MKK4, mitogen-activated protein kinase 4; PBS, phosphate buffered saline; PHB1, prohibitin-1.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Activation Assay, Control, Expressing, Saline

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: A20 Coimmunoprecipitates with RIPK1 and ASK1 in vitro . A , HEK293T cells were cotransfected to overexpress HA-RIPK1 and A20-Flag for 24 h. Cells were harvested and IP was performed with HA Tag, Flag Tag, or isotype matched control IgG. Membranes were immunoblotted for A20 and RIPK1. B , HEK293T cells were cotransfected to overexpress the kinase dead mutant HA- RIPK1 D138N and A20-Flag for 24 h, and subsequently lysates were IPed with HA Tag, Flag Tag, or isotype control IgG. Membranes were immunoblotted for A20 and RIPK1. C , HEK293T cells were cotransfected to overexpress GFP-A20 and ASK1-Flag for 24 h, and subsequently IP was performed with GFP Tag, Flag Tag, or isotype control IgG. Membranes were immunoblotted for A20 and ASK1. D , HEK293T cells were cotransfected to overexpress ASK1-Flag and HA-RIPK1 or control IgG for 24 h. Lysates were IPed with HA tag and immunoblotted against ASK1and RIPK1. Results of at least three independent experiments.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: In Vitro, FLAG-tag, Control, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: RIPK1 interferes with the A20-ASK1 interaction by binding to and sequestering A20 in vitro . A , HEK293T cells were cotransfected to overexpress GFP-A20 and ASK1-Flag either with HA-RIPK1 or control vector for 24 h. Cells were harvested and IP was performed with GFP Tag or isotype-matched control IgG. Membranes were immunoblotted for ASK1, A20, and RIPK1. B , HEK293T cells were cotransfected to overexpress GFP-A20 and ASK1-Flag either with HA-RIPK1 or control vector for 24 h, and subsequently lysates were IPed with Flag Tag or isotype-matched control IgG. Membranes were immunoblotted for A20, ASK1, and RIPK1.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Binding Assay, In Vitro, Control, Plasmid Preparation, FLAG-tag

Journal: The Journal of Biological Chemistry

Article Title: Interaction of RIPK1 and A20 modulates MAPK signaling in murine acetaminophen toxicity

doi: 10.1016/j.jbc.2021.100300

Figure Lengend Snippet: Proposed signaling mechanism. After APAP, ASK1 is activated (pASK1) and results in the activation of MKK4 (pMKK4), pJNK, and liver injury . Knockout of RIPK1 in hepatocytes leads to increased A20 levels, which allow for A20 to bind, sequester, and possibly inactivate or degrade ASK1, thus yielding a downregulation of pMKK4 and pJNK, which ultimately result in an attenuation in liver injury. Conversely, in hepatocyte-specific A20 knockout livers, ASK1 protein is increased, leading to phospho-activation of MKK4 and sustained pJNK activation, which result in increased liver injury.

Article Snippet: Human RIPK1 was purchased from Addgene (clone # 78,842) and was subsequently tagged with three C-terminal HA tags and subcloned into pCDNA 3.3 using a TOPO TA cloning kit (Thermo Fisher Scientific) according to manufacturer's protocols to yield our 3HA-pCDNA3.3-RIPK1 construct.

Techniques: Activation Assay, Knock-Out