Journal: bioRxiv

Article Title: Early Proteasome Gene Downregulation And Impaired Proteasomes Function Underlie Proteostasis Failure In Alzheimer’s Disease

doi: 10.1101/2025.01.21.634128

Figure Lengend Snippet: ( A–D ) Western blot analysis of Nrf1, PSMG1 and Rpt5/PSMC3 in proteasome-enriched soluble cortical extracts from control and AD brains (control n = 9, AD n = 9). ( A ) Representative immunoblots. Actin serves as a loading control. ( B–D ) Quantifications show that ( B ) total Nrf1 levels are significantly elevated in AD, while ( C ) PSMG1 is markedly reduced and ( D ) Rpt5/PSMC3 levels are not significantly reduced. ( E–G ) Subcellular fractionation of cortical extracts to assess Nrf1 localization. ( E ) Representative blots of total, cytosolic, and nuclear fractions from control and AD brains (control n = 15, AD n = 15). Lamin A/C and GAPDH are used as nuclear and cytosolic markers, respectively, and actin as a loading control. ( F,G ) Quantifications of upper and lower Nrf1 bands (corresponding to different post-translationally modified forms) show that total Nrf1 levels were unchanged between groups whereas the cytosolic levels were increased in AD. Contrary to the cytosolic Nrf1, nuclear Nrf1 is significantly decreased in AD. This suggests impaired nuclear translocation or processing of Nrf1 required for effective transcriptional activation of proteasome genes. (I) Representative Western blots showing Nrf1, PSMG1 and Rpt5 in total lysates, cytosolic, and nuclear fractions of two cell lines (DS1 and DS9) treated with or without epoxomicin, a proteasome inhibitor (four biological experiments). Lamin A/C serves as a nuclear marker, GAPDH as a cytosolic marker, and β-actin as a loading control. (B–D) Quantifications of ( B ) Nrf1, ( C ) PSMG1, and ( D ) Rpt5 levels comparing DS9 to DS1, a control condition. In the absence of epoxomicin, Nrf1 (upper and lower bands) undergoes rapid degradation, maintaining low basal levels (DS1cells, control condition). Upon reduced proteasome activity under persistent tau aggregation (DS9 cells condition) Nrf1 upper and lower bands increase in all the fractions. Upon proteasome inhibition with epoxomicin, cytosolic Nrf1 accumulate in the nucleus, indicative of the activated “bounce-back” response aimed at restoring proteasome capacity. This response includes upregulation of PSMG1 in both total and nuclear fractions. Rpt5 levels also show modest changes. These results demonstrate that pharmacological proteasome inhibition can recapitulate aspects of the compensatory mechanism attempting to restore proteasome homeostasis and highlight the enhanced responsiveness in a proteostasis-compromised cell line (DS9). Data are presented as mean ± SEM; each point represents an individual sample; ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Antibody dilutions for Western Blots are as follows: rabbit monoclonal anti-Nrf1 (1:1,000, 8052, Cell Signaling Technologies), rabbit polyclonal anti-PSMG1/PAC1 (1:1,000, 13378, Cell Signaling Technologies), mouse polyclonal anti-Rpt5/PSMC3 (1:2500, BML-PW8770, Enzo Life Sciences), mouse monoclonal anti-Lamin A/C (1:4,000, 4777, Cell Signaling Technologies), mouse monoclonal anti-GAPDH (1:8,000, 97166, Cell Signaling Technologies), mouse monoclonal anti-β-Actin (1:8,000, 3700, Cell Signaling Technologies).

Techniques: Western Blot, Control, Fractionation, Modification, Translocation Assay, Activation Assay, Marker, Activity Assay, Inhibition

Journal: Neurochemical Research

Article Title: Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation

doi: 10.1007/s11064-023-03989-7

Figure Lengend Snippet: List of primary antibodies applied for immunohistochemistry

Article Snippet: anti-PAC1 receptor , rabbit polyclonal , 1:500 , ThermoFisher Scientific, Budapest Hungary (PA3-115).

Techniques: Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Relative pituitary adenylate cyclase-activating peptide receptor (PAC1R) mRNA transcript abundances in the myometrial layer of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups, estimated by real-time PCR. Relative PAC1R mRNA transcript abundances are expressed as the mean ± SEM of ratios in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques: Control, Saline, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Relative pituitary adenylate cyclase-activating peptide receptor (PAC1R) protein abundances in the myometrial layer of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups, estimated by Western blot analysis. The relative PAC1R protein abundances are expressed as the mean ± SEM of ratios in relation to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The blot with representative bands for each group is presented in . * p < 0.05, *** p < 0.001 compared between groups.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques: Control, Saline, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Representative pictures show pituitary adenylate cyclase-activating peptide receptor (PAC1R) immunostaining in the myometrial layer of gilts from the control (CON), saline (SAL) and E. coli ( E. coli ) groups. Positive immunoreaction to PAC1R is visible in muscle cells and arteries (endothelium, muscle layer) of the myometrium of the control ( A ), saline-injected ( B ) and inflamed ( C ) uteri. Negative control (NC) for PAC1R ( D ) was obtained by omitting the primary antibody. MMC—myometrial muscle cells; A—artery. The scale bar of each image is 50 µm in length.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques: Immunostaining, Control, Saline, Injection, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Influence of pituitary adenylate cyclase-activating peptide (PACAP) alone ( A , C ) and PACAP receptor (PAC1R) antagonist with PACAP ( B , D ) on the contractile amplitude in the myometrium ( A , B ) and endometrium/myometrium ( C , D ) strips of gilts from the CON (grey bars), SAL (hatched bars) and E. coli (black bars) groups. Results were calculated for five gilts in each group. The actions of the antagonist (a dose of 10 − 6 M) and particular PACAP doses are depicted as percentage (mean ± SEM) changes from the basal (pre-treatment period) amplitude taken as 100% (horizontal lines). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the basal value in each group; A p < 0.05, AA p <0.01, AAA p <0.001 between the CON and E. coli groups for the same treatment; BB p < 0.01, BBB p < 0.001 between the SAL and E. coli groups for the same treatment; C p < 0.05 between the CON and SAL groups for the same treatment; # p <0.05, ## p < 0.01, ### p < 0.001 between the antagonist with PACAP action versus PACAP action alone for the same group/tissue/PACAP dose.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Investigation of the Role of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and Its Type 1 (PAC1) Receptor in Uterine Contractility during Endometritis in Pigs

doi: 10.3390/ijms23105467

Figure Lengend Snippet: Influence of pituitary adenylate cyclase-activating peptide (PACAP) alone ( A , C ) and PACAP receptor (PAC1R) antagonist with PACAP ( B , D ) on the contractile frequency in the myometria ( A , B ) and endometrium/myometrium ( C , D ) strips of gilts from the CON (grey bars), SAL (hatched bars) and E. coli (black bars) groups. Results were calculated for five gilts in each group. The actions of the antagonist (a dose of 10 − 6 M) and particular PACAP doses are depicted as the percentage (mean ± SEM) change from the basal (pre-treatment period) frequency, taken as 100% (horizontal lines). * p < 0.05, ** p < 0.01, *** p < 0.001 compared to the basal value in each group; A p < 0.05, AAA p < 0.001 compared between the CON and E. coli groups for the same treatment; B p < 0.05, BBB p < 0.001 compared between the SAL and E. coli groups for the same treatment; # p < 0.05, ## p < 0.01, ### p < 0.001 compared between the antagonist with PACAP action versus PACAP action alone for the same group/tissue/PACAP dose.

Article Snippet: To block the non-specific bindings, membranes were incubated with 5% fat-free dry milk in a TBS-T buffer at 21 °C for 1.5 h. They were then incubated at 4 °C for 18 h with primary PAC1 receptor polyclonal rabbit antibody (dilution: 1:1000, cat. no. CBS-PA207627, Cusabio Biotech Co.).

Techniques:

Journal: European Journal of Histochemistry : EJH

Article Title: Secreted key regulators (Fgf1, Bmp4, Gdf3) are expressed by PAC1-immunopositive retinal ganglion cells in the postnatal rat retina

doi: 10.4081/ejh.2022.3373

Figure Lengend Snippet: List of primary antibodies used for immunohistochemistry.

Article Snippet: anti-PAC1 receptor , Rabbit polyclonal , 1:1000 , ThermoFisher Scientific, Budapest Hungary (PA3-115).

Techniques: Immunohistochemistry