cas 88899  (MedChemExpress)

Journal: Cells

Article Title: An Intrinsic Host Defense against HSV-1 Relies on the Activation of Xenophagy with the Active Clearance of Autophagic Receptors

doi: 10.3390/cells13151256

Figure Lengend Snippet: The degradation of autophagic receptors SQSTM1/p62, OPTN1, NBR1, and NDP52 during HSV-1 infection depends on the autophagic flux. H4 cells were either uninfected (mock) or infected with HSV-1 strain F at MOI 10. They were then treated with either vehicle DMSO (control) or 100 nM Baf A1 for 8 h to inhibit autophagic flux. Protein extracts were subjected to immunoblot using monoclonal antibodies against ( A ) SQSTM1/p62, OPTN1, NBR1, and NDP52. HSV-1 ICP8, an early protein, was used to control the infection. β-ACTIN was used as a loading control. Positions of molecular weight markers (kDa) are indicated on the left. Densitometric quantification of ( B , C ) SQSTM1/p62, ( D , E ) OPTN1, ( F , G ) NBR1, and ( H , I ) NDP52 protein levels were normalized to β-ACTIN. Statistical significance was determined by Student’s t -test. Bars represent means ± SD of biological replicates (SQSTM1/p62 n = 3; OPTN1 n = 3; NBR1 n = 3; NDP52 n = 3); n.s., not significant; * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The HALT protease and phosphatase inhibitor cocktail was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and RIPA Lysis Buffer, Dimethyl sulfoxide (DMSO; CAS 67-68-5), Z-Leu-Leu-Leu-al (MG132; CAS 133407-82-6), Chloroquine (CQ; CAS 50-63-5), and Bafilomycin A1 (Baf A1; CAS 88899-55-2) were purchased from Sigma Aldrich (St. Louis, MO, USA).

Techniques: Infection, Control, Western Blot, Molecular Weight

Journal: EMBO Reports

Article Title: Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

doi: 10.15252/embr.202153659

Figure Lengend Snippet: A Representative pseudo colour images of sypHy fluorescence in WT and Bsn GT hippocampal neurons at rest, upon stimulation with 40 and 900 APs at 20 Hz (in the presence of bafilomycin A1) and upon application of NH 4 Cl to visualise SVs that were refractory to electrical stimulation. B Average traces of the normalised fluorescence change (Δ F / F 0 ) of sypHy in WT (black) and Bsn GT (red) neurons as described in (A). Intensities were normalised to the peak of NH 4 Cl response. C Quantifications of mean RRP fraction in WT and Bsn GT . D Frequency distribution histograms of the response amplitudes of 1,050 individual synaptic puncta (from six independent experiments) to stimulation with 40 AP. The distribution is shifted to lower values in Bsn GT . E Quantifications of mean TRP fraction in WT and Bsn GT . F Frequency distribution histograms of synaptic response amplitudes in Bsn GT and WT neurons stimulated with 900 AP. Note the shift in distribution between genotypes. G Representative images of Syt1Ab uptake (magenta) driven by endogenous network activity in hippocampal neurons (18 DIV) from WT and Bsn GT mice. VGLUT1 (green) marks excitatory presynapses (upper panels) and VGAT the inhibitory ones (lower panels). H Quantification of normalised IF of Syt1Ab uptake in excitatory (red boxes) and inhibitory (pink boxes) synapses on experiments in (G). I Quantification of the fraction of active (Syt1Ab‐labelled) excitatory and inhibitory synapses on experiments illustrated in (G). J Representative images of Syt1Ab uptake (magenta) upon depolarization with 50 mM KCl. Identical cultures and staining were applied as in (G). K Quantification of normalised IF of Syt1Ab uptake on experiments from (J). L Quantification of the fraction of active excitatory and inhibitory synapses in (J). Data information: In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. In the frequency distribution histograms (D and F) black lines depict superimposed Gaussian fits for each group. The dashed lines in (H) and (K) depict IF in WT that were used for normalisation. The sample size n (in parentheses) corresponds to the number of analysed imaging experiments (B, C, E) or to the number of analysed images (G, J). In (D) and (F), the data of 1,050 synapses per genotype were processed. Data is obtained from 6 (A–F) or 3 (G–L) independent culture preparations. Significance was assessed with the Student's t ‐test; *** P < 0.001. Scale bar is 2 μm in (A) and 5 μm in (G) and (J).

Article Snippet: InSolution Roscovitine (CAS # 186692‐46‐6) and Bafilomycin A1 (CAS #88899‐55‐2) were from Calbiochem.

Techniques: Fluorescence, Activity Assay, Staining, Imaging

Journal: EMBO Reports

Article Title: Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

doi: 10.15252/embr.202153659

Figure Lengend Snippet: A, B Representative pseudo colour images (A) and average traces (B) of sypHy fluorescence plotted for WT and Bsn GT neurons treated with a CDK5 inhibitor roscovitine (rosc, 100 μM, grey and pink trace) or vehicle (black and red trace) for 30 min before stimulation with 40 and 900 APs in the presence of bafilomycin A. C, D Plots show mean values of RRP (C) and TRP (D) fraction for both genotypes before and after treatment. E Roscovitine treatment has a significantly higher effect on RRP and TRP in Bsn GT neurons compared with WT. Data information: n corresponding to the number of imaging experiments done on four independent cell preparations is given in brackets for each analysis. In the plots, the interquartile range and median are depicted as boxes, minimal and maximal values as whiskers, and + indicates mean. Significance was assessed by two‐way ANOVA with the Tukey's multiple comparison test (C, D) and by the Student's t ‐test (E) * P ≤ 0.05, ** P < 0.01, *** P < 0.001. Scale bar is 2 μm.

Article Snippet: InSolution Roscovitine (CAS # 186692‐46‐6) and Bafilomycin A1 (CAS #88899‐55‐2) were from Calbiochem.

Techniques: Fluorescence, Imaging, Comparison