Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure1. siRNA-mediatedIP3R1knockdownreducescalpainactivityandstabilizesAChRclustersinC2C12cells.A,Left,Trans- fection of two IP3R1-specific siRNA sequences to C2C12 (C2-siRNA) significantly reduced IP3R1 protein level to 0.07 0.01 of normal C2C12 (C2), while no difference was detected in control RNA-treated C2C12 (C2-ctRNA; 1.07 0.08) (#, normalized to untreated C2). Right, C2C12 cells, treated as indicated in the panels and immunostained with antibody to IP3R1 (green), show markedreductioninIP3R1immunostaininginC2C12cellstreatedwithIP3R1-specificsiRNAcomparedtonormalorctRNA-treated C2C12; n 3; **p 0.01, Student’s t test. All other comparisons are not significant. B, Time course of calpain activity (#, normalized to 0 time) in C2C12 cells treated with the following: media alone (C2); carbachol (CCh; 10 M); C2/CCh with 100 M calcium (Ca 2/CCh); C2/Ca 2/CCh after control RNA (Ca 2/CCh/ctRNA); Ca 2/CCh after siRNA-mediated IP3R1 knockdown (Ca 2/CCh/siRNA);Ca 2/CChwith10Mxestospongin(Ca 2/CCh/XS);andCa 2/CChwith50M2-APB(Ca 2/CCh/2-APB). Aftertreatmentfor60min,calpainactivityforCCh,Ca 2/CCh,andCa 2/CCh/ctRNAwas2.180.11,2.770.21,and2.74 0.18-fold over untreated C2, respectively. After pretreatment of C2 with siRNA-IP3R1, XS, and 2-APB, carbachol-induced calpain activity was 1.31 0.79 (Ca 2/CCh/siRNA), 1.33 0.10 (Ca 2/CCh/XS), and 1.38 0.10 (Ca 2/CCh/2-APB), respectively, significantlylessthancontrolnotpretreated;n3,*p0.05,Mann–WhitneyUtest.Allothercomparisonsarenotsignificant. C, Top, AChR clusters expressed in untreated and treated C2C12 cultures. AChR clusters labeled with TxR-BT were prominent in C2, Ca 2/CCh/siRNA, Ca 2/CCh/XS, and Ca 2/CCh/2-APB and visibly reduced in CCh, Ca 2/CCh, and Ca 2/CCh/ctRNA, Scale bar, 40 m. Bottom, AChR cluster density [#, high-powered field (HPF)] in cultures untreated (C2, 575.742.7) and treated for

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Control, Activity Assay, Knockdown, Labeling, Immunofluorescence, Western Blot

Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure 2. Reduced expression IP3R1 in tibialis anterior and neuromuscular junction by siRNA-mediated IP3R1 knockdown. A, Left, Transfection TA muscle with GFP-specific siRNA sequence and GFPplasmidsignificantlyreducedGFPproteinlevelto0.160.01ofGFP-electroporatedTAmuscleonday5,whilenodifferencewasdetectedincontrolRNA-treated,GFP-transfectedTAmuscle (ctRNA-5 d; 1.04 0.09). Fifteen days after electroporation of TA with GFP-siRNA and GFP plasmid (siRNA,15 d), GFP protein level returned to 0.95 0.03 of GFP-plasmid transfected TA (#, normalized to only GFP-expressed TA muscle on days 5). Right, TA muscle treated as indicated in panels and compared to normal GFP-expressed TA muscle. GFP fluorescence showed remarkable reductioninTAmuscletreatedwithGFP-specificsiRNA;n3;*p0.01,Student’sttest.Allothercomparisonsarenotsignificant.B,ElectroporationoftwoIP3R1-specificsiRNAsequences(S1and S2) into TA muscle diminished IP3R1 expression maximally between days 7 and 9. IP3R1 protein level (#, normalized to untreated) was reduced to 0.11 0.02 and 0.15 0.02% of untreated on days7and9,respectively,andrecovered0.990.06%onday14;n9,**p0.001,Mann–WhitneyUtest.Allothercomparisonsarenotsignificant.C,IP3R1immunostainedwithantibodyto IP3R1(green)colocalizeswithNMJlabeledwithTxR-BT(red)andsurroundedbysubsynapticDAPI-labelednuclei(blue)inWTandctRNA-treatednormalTAmuscle.ExpressionofIP3R1atNMJwas nearly eliminated in all nine TA muscles electroporated with IP3R1 siRNA. Scale bar, 12 m. D, Immunostaining of IP3R1 (green) does not colocalize with synaptophysin (red), a subsarcolemmal region, and does not colocalize with S100 protein (red, middle right panel), When labeled for AChRs with TxR-BT, muscle electroporated with GFP-ctRNA shows the RNA in the subsarcolemmal region surrounding the TxR-BT-labeled endplates (red, far right panel). Scale bar, 12 m.

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Expressing, Knockdown, Transfection, Sequencing, Electroporation, Plasmid Preparation, Fluorescence, Muscles, Immunostaining, Labeling, Immunofluorescence, Western Blot

Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure 3. siRNA-mediated IP3R1 silencing alters presynaptic and postsynaptic components of neuromuscular transmission. A. Left, Representative traces of MEPCs in ctRNA-treated (top, black)andIP3R1-siRNA-treated(bottom,blue)normaltibialisanteriormusclevoltageclamped at 60 mV. Right, The frequency of spontaneous MEPC was significantly increased in IP3R1- siRNA-treatedmusclecomparedwithctRNA-treatedTA(n29)(3.110.26s 1;n49vs 1.4 0.09 s 1; n 29, **p 0.001). B, MEPC amplitudes from IP3R1-siRNA-treated TA muscle (3.86 0.32 nA; n 25) were nearly 2-fold greater than those recorded from ctRNA- treatedTAmuscle(2.530.29nA;n19,*p0.05).C,TheMEPC( )inIP3R1-siRNA-treated TA muscle (1.5 0.08 ms; n 25) was significantly increased compared with that of ctRNA (1.140.15ms;n19,*p0.05).D,EPCamplitudeofIP3R1-siRNA-treatedTAmusclewas significantgreaterthanthatofthectRNA-treatedmuscle(50.215.9nA;n26vs13.72 2.8 nA; **p 0.001, n 26). E, The quantal content of evoked transmitter release was significantlyincreasedinIP3R1-siRNA-treatedTAmusclecomparedwithctRNA-treatedmuscle (34.21.8vs26.083.96,*p0.05)andincreasedby30%(n26).Insetshowsrawdata traceofEPCforIP3R1-siRNA-treatedandctRNA-treatedmuscle;n#ofendplates.Mouse# 10.AllcomparisonswereanalyzedbyStudent’sttest.Allothercomparisonsarenotsignificant.

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Transmission Assay, Patch Clamp

Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure 4. IP3R1 activity controls synaptic gene expression in muscle. A, AChR intensity (TxR-BT fluorescence intensity; #, normalizedtountreated)atWTNMJaftertreatmentwithsiRNA(siRNA-WT)forIP3R1knockdown(9d)is1.760.18-foldgreater than untreated, a significant increase compared with untreated (n 7; ***p 0.001), while ctRNA (ctRNA-WT) had no effect (n 7; p 0.81). After denervation (Dv), the AChR intensity was increased by 2.79 0.58-fold, significantly greater than untreated (n 7; ***p 0.001). B, Time course of expression (#, normalized to 0 time expression) of and subunit after denervation (left graph), or after siRNA silencing of IP3R1 (right graph). Expression levels at day 9 after siRNA for subunit (11.12 1.83) and subunit (63.48 6.49) were significantly greater than the corresponding values for ctRNA-treated TA muscle(n5;p0.005).C,Expressionlevels(#,normalizedto0timeexpression)ofNMJgenes,AChR subunit(delta),AChR subunit (), acetylcholinesterase (AChE), ErbB2, MuSK, and rapsyn in TA muscle. Expression levels at day 9 after siRNA for subunit(3.070.64;**p0.005)andsubunit(1.520.16;**p0.005)weresignificantlygreaterthanthecorresponding valuesforctRNA-treatedTAmuscle(n5).Treatmentwithdenervationfor3d(DV-3d-WT),IP3R1knockdownfor9d(siRNA-9d- WT), combined denervation and IP3R1 knockdown (DV-3d-siRNA-9d-WT), or ctRNA for 9 d (ctRNA-WT) showed similar but not identical effects of the two treatments on gene expression. Combined treatments showed that effects are overlapping. ***p 0.001.D,Expressionlevels(#,normalizedto0timeexpression)ofmusclegenes,Glut1andGlut4,inTAmuscleaftertreatmentwith denervation for 3 d (DV-3d-WT), IP3R1 knockdown for 9 d (siRNA-9d-WT), combined denervation and IP3R1 knockdown (DV-3d- siRNA-9d-WT), or ctRNA for 9 d (ctRNA-WT) showed that IP3R1 had no effect on non NMJ muscle gene expression compared with denervation.ComparedtoWT,expressionofGlut1/4wasunchangedinsiRNA-9d-WTandctRNA-9d-WT,whereasGlut1expression was 15-fold to WT in Dv-3d-siRNA-9d-WT and in Dv-3d-WT. ctRNA had no effect on gene expression; n 5; **p 0.005, ***p 0.001. E, Expression levels (#, normalized to 0 time expression) of regulatory genes, myogenin, Dach2, and HCAC4 in TA muscleaftertreatmentwithdenervationfor3d(DV-3d-WT),IP3R1knockdownfor9d(siRNA-9d-WT),combineddenervationand IP3R1knockdown(DV-3d-siRNA-9d-WT),orctRNAfor9d(ctRNA-WT)demonstratedthatcomparableeffectsonregulatorygenes was significantly increased by IP3R1 knockdown and denervation. ctRNA had no effect on gene expression; n 5; **p 0.005, ***p 0.001. F, Left, Immunolocalization of HDAC4 in subsynaptic nuclei of wild-type (WT), denervated (Dv), siRNA-treated (siRNA), and control RNA-treated (ctRNA) TA muscle colabeled with TxR-BT (red) and DAPI (blue) to label nuclei shows that HDAC4(green)islocalizedatandsurroundingNMJsinallfourpanels,butinadditionshowscolocalizationwithsubsynapticnuclei (light blue stain) in denervated and siRNA-treated TA muscle (arrows). Scale bar, 15 m. Right, Quantification of HDAC4 immu- nolabeling in subsynaptic nuclei (SN) and extrajunctional nuclei (EN) showed that the percentage of HDAC4-labeled subsynaptic nuclei(HDAC4-SN)indenervatedTAmuscle(Dv-3d-WT)andsiRNA-9d-WTwere12.070.73and7.360.29%,respectively,

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Activity Assay, Gene Expression, Fluorescence, Expressing, Knockdown, Control, Staining, Labeling, CRISPR

Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure 6. Decremental responses in rsEMG in SCS transgenic mouse and neostigmine my- opathy depend on IP3R1 activity. Top, Compound muscle action potentials recorded during repetitive stimulation (rsEMG) in WT, neostigmine myopathy (Neo-WT), SCS, and SCS treated with siRNA to knockdown IP3R1. Decremental responses are seen in both SCS and Neo-WT. Bottom, Quantitation of decrement in rsEMG in WT, neostigmine myopathy, and SCS under indicated conditions. Decrement in WT rsEMG is only 2.06 1.28% but is significantly in- creasedinneostigminemyopathy(Neo-WT,10.551.12%)andSCS(14.751.37%).IP3R1 silencingbysiRNAreducesdecrementto5.550.73%inneostigminemyopathyand7.48 1.14% in SCS. ctRNA had no effect in either condition. Blockade of IP3R1 by 2-APB reduced decrementto8.002.07%,whilesalinehadnoeffect;n5;*p0.005,Mann–WhitneyU test. All other comparisons not significant.

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Transgenic Assay, Activity Assay, Knockdown, Quantitation Assay

Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure 7. siRNA-mediated IP3R1 silencing alters SCS presynaptic and postsynaptic compo- nentsofneuromusculartransmission.A,Left,RepresentativetracesofMEPCsinctRNA-treated andsiRNA-treatedSCSmousetibialisanteriormuscleendplates,voltageclampedat60mV. Right, The frequency of spontaneous MEPC was significantly increased in IP3R1-siRNA-treated muscle (n 20) compared with ctRNA-treated TA (n 19) (1.75 0.1 s 1 vs 0.78 0.04 s 1, ***p 0.001). B, MEPC amplitudes from IP3R1-siRNA-treated SCS TA muscle (1.63 0.19,n20)werenearly47%greaterthanthoserecordedfromctRNA-treatedSCSTAmuscle (1.11 0.12, n 10; *p 0.03). C, The MEPC ( ) in IP3R1-siRNA-treated TA (14.04 2.89 ms, n 13) remained prolonged and unchanged compared with that of ctRNA-treated SCS, consistent with the presence of the SCS mutation L269F (13.72 2.85 ms, n 7). D, EPC amplitudeofIP3R1-siRNA-treatedTAmuscle(n13)wassignificantlygreaterthanthatofthe ctRNA-treated muscle (45.59 6.45 nA; n 13 vs 13.72 2.8 nA; n 7; **p 0.003). E, The computed quantal content of evoked transmitter release was nearly doubled in IP3R1- siRNA-treated SCS TA muscle compared with ctRNA-treated muscle (28.3 4.6; n 13 vs 12.552.32;n7,*p0.03).InsetshowsaveragetracesofMEPCandEPCforIP3R1-siRNA- treated and ctRNA-treated SCS endplates; n # of endplates. Mouse # 4. All comparisons were analyzed by Student’s t test. All other comparisons not significant.

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Mutagenesis, Patch Clamp, Knockdown

Journal: Journal of Neuroscience

Article Title: Skeletal Muscle IP3R1 Receptors Amplify Physiological and Pathological Synaptic Calcium Signals

doi: 10.1523/jneurosci.3766-11.2011

Figure Lengend Snippet: Figure 8. IP3R1 silencing improves motor function in SCS mice. A, Hindlimb motor time interval parameters measured in motorized treadmill (DigiGait) in WT and SCS mice and the effect of unilateral IP3R1 silencing. Functional parameters termed “swing”, “brake”, and “pro- pel” are time intervals for three components of normal leg movements recorded during walk- ing;asdefinedinMaterialsandMethods,theseareidenticalbetweenleftandrighthindlimbin WTmice.AllthreeparametersaresymmetricallyandsignificantlyincreasedinSCSmice.siRNA treatment of left hindlimb TA causes significant improvement in each of these parameters in comparison to the contralateral right hindlimb, which was treated with ctRNA; n 11; *p 0.001, Student’s t test. All other comparisons not significant. B, Mean hindlimb stride length andstepfrequencyinWTandSCSandtheeffectofunilateralIP3R1silencing.Theaveragestride length for both limbs in SCS is significantly longer than WT, while the step frequency is lower. siRNA treatment of the left TA of the SCS mice reduced the stride length to that of WT and increasedthestepfrequencytoslightlygreaterthanthatofWT;n11;*p0.001,Student’s t test. All other comparisons not significant.

Article Snippet: GFP-tagged control siRNA was purchased from Santa Cruz Biotechnology Primary antibodies were used against the following targets: neural filament (1:1000; Abcam), phosphorylated H2AX (Ser139; 1:500; Millipore), cleaved caspase 3 and 9 (1:200;Cell Signaling Technology), synaptophysin (1:500; Cell Signaling Technology), S100 (1:100; Abcam), histone deacetylase 4 (HDAC4; 1:500; Abcam), glial fibrillary acidic protein (1:500; Cell Signaling Technology), fibronectin (1:500; Abcam), IP3R1 (1:500; Alomone), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5000; Ambion).

Techniques: Functional Assay, Comparison

Journal: bioRxiv

Article Title: Flaviviruses alter endoplasmic reticulum-mitochondria contacts to regulate respiration and apoptosis

doi: 10.1101/2023.03.09.531853

Figure Lengend Snippet: (a) Schematic representation of ERMC tethering complexes. Generated with BioRender. (b) Huh7.5 cells were infected with DENV 16681s (MOI=1) or ZIKV H/PF/2013 (MOI=10) or left uninfected. Two or three days later, cells were fixed and subjected to proximity ligation assays (PLA) to detect SYNJ2BP-RRBP1 interactions, and immunostained for NS3 viral protein to identify infected cells. Cells were imaged using confocal microscopy. Representative images are shown. Scale bar=20 µm. (c) Quantification of PLA dot abundance for RRBP1-SYNJ2BP, VAPB-PTPIP51 and IP3R1-VDAC1 interactions in uninfected-and infected-cells from two independent experiments. Values in the 5%-95% percentile range are shown. (d) Cells were prepared as in (b). Two or three days post-infection, cell extracts were prepared, and the expression levels of the indicated proteins were analyzed by western blotting. The arrow indicates the ZIKV-induced SYNJ2BP protein product.

Article Snippet: Rabbit anti-IP3R1 (NBP2-22458) antibodies were obtained from Novus Biologicals.

Techniques: Generated, Infection, Ligation, Confocal Microscopy, Expressing, Western Blot

Journal: bioRxiv

Article Title: Flaviviruses alter endoplasmic reticulum-mitochondria contacts to regulate respiration and apoptosis

doi: 10.1101/2023.03.09.531853

Figure Lengend Snippet: (a) Fixed Huh7.5 cells stably expressingmito-mTurquoise2 (exhibiting fluorescent mitochondria) were subjected to PLAs detecting VDAC1-IP3R1, SYNJ2BP-RRBP1 and VAPB-PTPIP51 interactions. Scale bar = 10 µm. (b,c) Huh7.5 cells were infected with DENV 16681s (MOI=1) or ZIKV H/PF/2013 (MOI=10), or left uninfected. Two days later, cells were fixed and subjected to proximity ligation assays (PLA) to detect (b) VDAC1/IP3R1 or (c) PTPIP51/VAB interactions, and immunostained for NS3 viral protein to identify infected cells. Cells were imaged using confocal microscopy. Representative images are shown.

Article Snippet: Rabbit anti-IP3R1 (NBP2-22458) antibodies were obtained from Novus Biologicals.

Techniques: Stable Transfection, Infection, Ligation, Confocal Microscopy

Journal: Nature

Article Title: Inositol-1,4,5-trisphosphate receptor regulates hepatic gluconeogenesis in fasting and diabetes.

doi: 10.1038/nature10988

Figure Lengend Snippet: Figure 3 | Glucagon stimulates CRTC2 activity via PKA-dependent phosphorylation of InsP3Rs. a, b, Immunoblots of InsP3R1 immunoprecipitates (IP) using phospho-PKA substrate antiserum to show effect of H89 (a) and Ala mutations (b) at one or both (DM) PKA consensus sites (Ser 1589, Ser 1756) on InsP3R1 phosphorylation in hepatocytes exposed to glucagon (Gcg). Effect of wild-type and PKA-mutant InsP3R1 on calcium mobilization in response to Gcg (b) shown (*P , 0.001; n 5 3). c, d, Effect of wild-type or PKA-defective InsP3R1 (DM) on calcineurin (Calna) activation

Article Snippet: The antibodies anti-InsP3R1 (A302–158A) and InsP3R3 (A302–160A) were purchased from Bethyl Laboratories, anti-InsP3R2 (ab77838) from Abcam, anti-calcineurin (610260) from BD Biosciences, antiGRP78 (ADI-SPA-826-F) from Enzo Life Sciences, anti-phospho-PKA substrate (RRXS/T, 9624), anti-phospho-AKT substrate (RXXS/T, 9614) and CRTC2 (pS171, 2892) from Cell Signaling.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, Mutagenesis, Activation Assay

Journal: MedComm

Article Title: cZFP609 Tethering BiP Alleviates Cartilage Degradation in Osteoarthritis via Remedying Aberrant ER‐Mitochondrial Contacts

doi: 10.1002/mco2.70405

Figure Lengend Snippet: cZFP609 reduces ERMCs via inhibiting the depolymerization of BiP. (A) Representative images of the ERMCs probed with MitoTracker and ER‐resident protein IP3R in chondrocytes treated with the indicated conditions by confocal microscopy and quantified by the Fiji image macro tool. Scale bar = 20 µm. (B) Representative TEM images of ERMCs in chondrocytes treated with the indicated conditions. Mitochondria and ER masks were manually segmented and distinguished in blue and yellow, respectively. ERMCs (distances between 100 and 30 nm) were quantified by the Fiji image macro tool. Scale bar = 500 nm. (C) Native PAGE analysis of BiP in chondrocytes treated with ATP in different concentrations. (D) Native PAGE analysis of BiP in cZFP609 overexpressed chondrocytes exposed to 150 µM ATP after being treated with TNFα (20 ng/mL). (E) Representative images of ERMCs in cZFP609 overexpressed chondrocytes exposed to 150 µM ATP after being treated with TNFα (20 ng/mL), and quantified by the Fiji image macro tool. Scale bar = 20 µm. (F) Representative co‐localization images and relative quantification by image Fiji macro tool in chondrocytes stained with MitoTracker and BiP. Scale bar = 20 µm. Data and images represent at least three independent experiments by paired t ‐test. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus the corresponding control.

Article Snippet: The membranes were blocked in 5% milk for 1 h at room temperature and then incubated with primary antibodies against MMP13 (1:1000, 18165‐1‐AP, Proteintech), Col2ɑ (1:1000, 28459‐1‐AP, Proteintech), Sox9 (1:200, sc‐166505, Santa Cruz), p53 (1:1000, 10442‐1‐AP, Proteintech), p16 (1:1000, A0262, ABclonal), p21(1:1000, HA500005 , HUABIO), Sirt1 (1:500, A11267, ABclonal), IL‐1β (1:500, 16806‐1‐AP, Proteintech), BiP (1:1000, 11587‐1‐AP, Proteintech), IRE1α (1:1000, 27528‐1‐AP, Proteintech), IRE1α‐p (1:1000, AP0878, ABclonal), XBP‐1S (1:1000, 24868‐1‐AP, Proteintech), ATF4 (1:1000, 60035‐1‐Ig, Proteintech), IP3R (1:1000, A4436, ABclonal), TFRC (1:1000, A5865, ABclonal), GPX4 (1:1000, A11243, ABclonal), FTH (1:1000, 11682‐1‐AP, Proteintech), ACSL4 (1:1000, 22401‐1‐AP, Proteintech), COX2 (1:1000, ET1610‐23, HUABIO), and GAPDH (1:2000, AC036, ABclonal) at 4°C overnight.

Techniques: Confocal Microscopy, Clear Native PAGE, Quantitative Proteomics, Staining, Control