anti rabbit zdhhc9  (Novus Biologicals)

Journal: bioRxiv

Article Title: ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

doi: 10.1101/2023.03.07.531538

Figure Lengend Snippet: ( A ) Click chemistry screen by rhodamine labeling in 293T cells co-expressing the 23 human zDHHCs with GSDMD to identify zDHHCs that enhanced GSDMD palmitoylation. Additional bands in the blots are likely from autopalmitoylation of zDHHCs. ( B ) Expression data of zDHHCs in HEK239T and THP-1 cells from Protein Atlas, identifying 5 highly expressed zDHHCs among those that enhanced GSDMD palmitoylation. ( C, D ) GSDMD-NT palmitoylation detected by ABE in HEK293T cells upon siRNA knockdown of the 5 highly expressed zDHHCs or a scrambled siRNA (scRNA) as a control (C), showing that knockdown of zDHHC5 and zDHHC9 compromised GSDMD-NT palmitoylation (D). ( E ) Anti-FLAG pulldown of zDHHS5 (left, catalytic mutant of zDHHC5) and zDHHC9 (right) by co-expressed GSDMD-FLAG. ( F-H ) PI positivity (F), LDH release (G), and viability (H) of GSDMD-NT expressing HEK293T cells upon siRNA knockdowns of zDHHCs. ( I ) Anti-FLAG immunofluorescence imaging of HEK293T cells with siRNA knockdowns of zDHHC5, zDHHC9, or both overexpressing GSDMD-NT-FLAG. Only cells treated with scRNA showed strong cell surface staining. Nuclei are marked by the DNA dye DAPI. ( J ) GSDMD palmitoylation detected by ABE in THP-1 cells upon siRNA knockdown of zDHHC5, zDHHC9, or both and treatment with LPS plus nigericin, showing that these knockdowns compromised GSDMD palmitoylation. ( K-M ) PI positivity (K), LDH release (L), and viability (M) of THP-1 cells upon siRNA knockdown of zDHHC5, zDHHC9, both, or other zDHHCs and treatment with LPS plus nigericin. All results were obtained from at least 3 independent experiments. Scale bars represent 5 μm (I). Error bars represent SEM. Statistics were measured by Student’s t-tests with *** for p<0.001, **** for p<0.0001, and NS (non-significant) for p>0.05.

Article Snippet: Immunoblots were probed with the following primary antibodies: 1:1000 rabbit anti-GSDMD monoclonal antibody (Cell Signaling Technology, 39754), 1:1000 rabbit anti-GSDMD polyclonal antibody (Novus Biologicals, NBP-33422), 1:500 rabbit anti-zDHHC5 polyclonal antibody (Proteintech, 21324-1-AP), 1:500 rabbit anti-zDHHC9 polyclonal Antibody (ABclonal, A7977), 1:1000 rabbit anti-calnexin Antibody (Cell Signaling Technology, 2433S), or 1:5000 mouse anti-GAPDH monoclonal antibody (Proteintech, 60004-1-IG).

Techniques: Labeling, Expressing, Mutagenesis, Immunofluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation

doi: 10.1101/2023.03.07.531538

Figure Lengend Snippet: ( A ) Expression level of zDHHC5 and zDHHC9 in THP-1 cells treated with LPS, LPS plus nigericin, or LPS plus nigericin additionally treated with ROS modulators. No apparent changes were observed. ( B ) GSDMD palmitoylation detected by ABE in THP-1 cells pre-treated by PalmB (general inhibitor), or ML348 (APT1 inhibitor)/ML349 (APT2 inhibitor) together, or not, followed by addition of an ROS modulator, LPS and nigericin. PalmB or ML348/ML349 did not alter GSDMD palmitoylation under treatment of the ROS activator AMA, but significantly increased GSDMD palmitoylation under treatment of ROS quenchers NAC and MitoT. ( C-E ) PI staining (C), LDH release (D) and cell viability (E) of the same treatment as in (B). Scale bars in (C) represent 30 μm. All results were obtained from at least 3 independent experiments. Error bars represent SEM. Statistics were measured by Student’s t-tests with ** for p<0.01, *** for p<0.001, **** for p<0.0001, and NS (non-significant) for p>0.05.

Article Snippet: Immunoblots were probed with the following primary antibodies: 1:1000 rabbit anti-GSDMD monoclonal antibody (Cell Signaling Technology, 39754), 1:1000 rabbit anti-GSDMD polyclonal antibody (Novus Biologicals, NBP-33422), 1:500 rabbit anti-zDHHC5 polyclonal antibody (Proteintech, 21324-1-AP), 1:500 rabbit anti-zDHHC9 polyclonal Antibody (ABclonal, A7977), 1:1000 rabbit anti-calnexin Antibody (Cell Signaling Technology, 2433S), or 1:5000 mouse anti-GAPDH monoclonal antibody (Proteintech, 60004-1-IG).

Techniques: Expressing, Staining

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: PCR and immunoblotting analysis of Zdhhc9 expression in wild-type and mutant mice. (A) Agarose gel electrophoresis of end-point PCR products from WT and mutant brain cDNA samples. mRNA confirmation assay 1 (MA1) primers were designed to amplify a Zdhhc9 mRNA region of 283 bp from the 5′UTR to Exon 2. mRNA confirmation assay 2 (MA2) primers were designed to amplify a Zdhhc9 mRNA region of 154 bp from Exon 2 to Exon 3. z9v1 primers were designed to amplify a mRNA region of 139 bp from exon 5 to exon 7, and z9v2 primers were designed to amplify a mRNA region of 106 bp from exon 5 to exon 7. HyperLadder 100 bp ladder (Bioline) was used as a marker of DNA size. (B) Comparison of the average ΔCt values of WT and mutant mouse brain samples ( n = 3 WT, 3 mutant) for MA2 and z9v1 after normalisation against Tbp and Hprt1 reference genes. cDNA from WT and mutant mouse brain was amplified for 40 cycles using specific primers for the different targets (MA2, z9v1, Tbp and Hprt1 ) and SYBR Select Master Mix. Statistical analysis (unpaired t -test, Minitab version 17) revealed a significant effect of genotype for MA2- Tbp ( p = .042), MA2- Hprt1 ( p = .046) and z9v1- Hprt1 ( p = .037). p value for z9v1- Tbp was 0.075. (C) PCR products amplified from cDNA derived from mRNA extracted from WT and Zdhhc9 mutant mouse brain. Primers were designed to anneal to a region in exon 1 (see MAO F in for sequence of primer) and the 3′-UTR (3UTR R in for sequence of primer). HyperLadder 1 kb ladder (Bioline) was included as marker of DNA size. (D) Lysates from HEK293T cells transfected with HA-tagged zDHHCs were probed with antibodies against zDHHC9 ( top ) and HA ( bottom ). (E) Brain lysates from WT and mutant mice were probed with antibodies against zDHHC9 ( top ) and beta actin ( bottom ). Position of molecular weight markers is shown on the left of all immunoblots.

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Western Blot, Expressing, Mutagenesis, Agarose Gel Electrophoresis, Marker, Comparison, Amplification, Derivative Assay, Sequencing, Transfection, Molecular Weight

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: Comparison of wild-type and Zdhhc9 mutant mice in hanging wire and rotarod tests. (A) Average time spent by mice on the wire. Statistics were conducted using an unpaired t -test in Minitab version 17, t(31) = −3.3, p = .002 for effect of genotype on time spent on the wire. (B) Average time spent by mice on the rotarod. Statistics were conducted using an unpaired t -test in Minitab version 17, ns for genotype ( n = 20 WT, 14 mutant).

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Comparison, Mutagenesis

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: Comparison of wild-type and Zdhhc9 mutant mice in the open field test. Time spent by WT and mutant mice in the outer and inner sections of the test and their velocity during this time. (A–C) displays these parameters during the habituation phase of the test; (D–F) displays these parameters during the test time ( n = 14 mutant, 20 WT). Statistics were conducted using general linear model, repeated measures in SPSS version 22 with time bin as the within-subjects factor and genotype as the between-subjects factor; F(1,32) = 4.646, p = .039 for effect of genotype in time spent in inner and outer zones and F(1,32) = 3.44, p = .073 for effect of genotype on velocity during habituation. Moreover, F(2,64) = 32.573, p < .001 for effect of time bin on time spent in inner and outer zones during habituation. There was no interaction between time bin and genotype for the time spent in inner and outer zones, F(2,64) = 0.482, p = .482. During test time, p = ns (non-significant) for effect of genotype on time spent in inner and outer zones and velocity. Each time bin represents a period of 5 min.

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Comparison, Mutagenesis

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: Performance of WT and Zdhhc9 mutant mice in the elevated plus maze. Distance moved (A), time spent in closed arms (B), time spent in open arms (C), and time spent in final third of open arms (D) are shown (n = 20 WT, 14 mutant). Statistics were conducted using an unpaired t -test in Minitab version 17; t(22) = 2.49, p = .021 for effect of genotype on time spent in the open arms, t(29) = −2.84, p = .008 for effect of genotype on time spent in closed arms and t(24) = 2.74, p = .011 for effect of genotype on time spent in the final thirds of open arms.

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Mutagenesis

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: Comparison of acoustic startle and PPI response of wild-type and Zdhhc9 mutant mice. (A) Startle reactivity of WT and Zdhhc9 mutant mice. The startle response to a sound at a range of levels (dB) was measured. Statistics were conducted using general linear model, repeated measures in SPSS version 22; F(1,44) = 13.622, p = .001 for effect of genotype ( n = 24 WT, 22 mutant). (B) Mice received a pre-pulse at the levels (dB) shown. Following this, the startle response to a 120 dB sound was measured. Statistics were conducted using general linear model, repeated measures in SPSS version 22; p > .05 for effect of genotype (n = 24 WT, 22 mutant).

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Comparison, Mutagenesis

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: Performance of Zdhhc9 mutant and WT mice in the Morris water maze. Distance moved (A), latency (B) and mean velocity (C) during the 5 acquisition days of the experiment are shown. Statistics were conducted using general linear model, repeated measures in SPSS version 22 with experiment day as the within-subjects factor and genotype as the between-subjects factor; F(1,44) = 8.935, p = .005 for effect of genotype on distance moved, F(4,176) = 27.647, p < .001 for effect of day on distance moved while no interaction was found between day and genotype with F(4,176) = 1.020 and p = .399. F(1,44) = 9.677, p = .003 for effect of genotype on latency, F(4,176) = 23.499 and p < .001 for effect of day on latency while no interaction was found between day and genotype with F(4,176) = 0.868 and p = .484 ( n = 26 WT, 20 mutant). The effect of genotype on velocity was non-significant. (D) Time spent in quadrants during the first 30 s and last 30 s of the probe trial to assess reference memory are shown. Statistics were conducted using general linear model, repeated measures in SPSS version 22; F(1,44) = 49.573, p < .001 for effect of quadrant during the first 30 s and F(1,44) = 41.805, p < .001 for effect of quadrant during the last 30 s (n = 26 WT, 20 mutant). (E) Time spent to reach the visible platform for WT and Zdhhc9 mutant mice during the visual cue trial of Morris water maze. Statistics were conducted using unpaired t -test in Minitab version 17; t(42) = −0.92, p = ns for effect of genotype (n = 26 WT, 20 mutant).

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Mutagenesis

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: MRI analysis of brains from WT and Zdhhc9 mutant mice. (A) Coronal images from WT and mutant mouse brains after ex vivo MRI scan in a 9.4 Tesla magnet. (B and C) Box and whiskers graphs showing the various data points for the volume of corpus callosum and hippocampus. The whiskers indicate the maximum and minimum data points while the thick horizontal line indicates the median. (B) without normalisation; (C) with normalisation to total brain volume ( n = 3 WT, 3 mutant).

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Mutagenesis, Ex Vivo

Journal: Experimental Neurology

Article Title: Disruption of the Zdhhc9 intellectual disability gene leads to behavioural abnormalities in a mouse model

doi: 10.1016/j.expneurol.2018.06.014

Figure Lengend Snippet: Analysis of membrane association and S-acylation of H-Ras in whole brain and hippocampus from WT and Zdhhc9 mutant mice. (A) Membrane and cytosol fractions from whole brain homogenates were resolved by SDS-PAGE and transferred to nitrocellulose, and subsequently probed with GAPDH, Syntaxin and H-Ras antibodies. The left panel shows representative immunoblots (position of molecular weight markers is shown on the left), whereas the right panel shows quantified data for % membrane association. Statistical analysis using an unpaired t -test indicated that there was no significant difference in H-Ras membrane association between WT and mutant samples; p > .05 (n = 3 WT, 3 mutant). M: membrane fraction, C: cytosolic fraction. (B) Acyl-RAC samples from whole brain or hippocampi were resolved by SDS-PAGE and transferred to nitrocellulose, and subsequently probed with H-Ras antibody. The upper panel shows representative immunoblots (position of molecular weight markers is shown on the left), whereas the lower panel shows quantified data from whole brain or hippocampi. Statistical analysis using an unpaired t -test indicated that there was no significant difference in H-Ras S-acylation between WT and mutant samples; p > .05 (n = 3 WT, 3 mutant). TI: total input, UT: Unbound Tris treated fraction, BT: Bound Tris treated fraction, UH: Unbound HA treated fraction, BH: Bound HA treated fraction.

Article Snippet: Primary antibodies used for immunoblotting were as follows: mouse beta actin (Abcam, ab8226, 1:3000), rat HA (Roche, 1:1000), rabbit zDHHC9 (St John's Laboratory, STJ92709, 1:1000), rabbit H-Ras (Santa Cruz, SC-520, 1:300), rabbit GAPDH (Cell Signaling, 14C10, 1:1000), mouse syntaxin (Sigma, HPC-1, 1:1000).

Techniques: Membrane, Mutagenesis, SDS Page, Western Blot, Molecular Weight