Journal: Communications Biology

Article Title: Immunoproteasome remodeling in senescing human macrophages reveals the loss of PA28αβ capping as a hallmark of immunosenescence

doi: 10.1038/s42003-025-08765-7

Figure Lengend Snippet: Representative western immunoblot analysis of proteasome (panα, β1, β2, and β5), immunoproteasome (β1i, β2i, and β5i) and regulatory particle (Rpt4 of the 19S RP and PA28β of the PA28αβ RP) subunit expression levels. Two micrograms of total protein was separated by SDS‒PAGE on a 12% (w/v) acrylamide gel. The total protein content in each lane was detected using No Stain reagent as a loading control. Immunoreactive bands and total protein signals were imaged with a ChemiDoc system and quantified with Image Lab software. The densitometric values of the immunoreactive bands were normalized to the total protein content and are reported in the graphs as the fold change vs day 7. Data are the mean ± S.D. * p < 0.05 and *** p < 0.005.

Article Snippet: The membranes were blocked and then incubated overnight (o/n) at 4 °C with the following primary antibodies (1:1000 dilution): anti-α1,2,3,5,6,7 (#PW8195, pan α) from Enzo Life Sciences; anti- PSMB6 /β1 (#A4053), anti- PSMB7 /β2 (#14771), anti- PSMB10 /β2i (#A5452), anti-19S PSMC6 /Rpt4 (#A5377), anti- PSME2 /PA28β (#A5562), anti-p16 INK4a (#A11651) from ABclonal; anti- PSMB8 /β5i (#13635), anti-p21 Waf1/Cip1 (#2355), anti-LC3B (#2775), anti-PCNA (PC10) (#2586) from Cell Signaling Technology; anti- PSMB9 /β1i (C-term) (#AP21207b) and anti- PSMB5 /β5 (# ALS17241 ) from Abcepta; anti-SQSTM-1/p62 (A302-856A-M) from Bethyl; and anti-Ubiquitin kindly provided by Prof. A.L.

Techniques: Western Blot, Expressing, Acrylamide Gel Assay, Staining, Control, Software

Journal: Communications Biology

Article Title: Immunoproteasome remodeling in senescing human macrophages reveals the loss of PA28αβ capping as a hallmark of immunosenescence

doi: 10.1038/s42003-025-08765-7

Figure Lengend Snippet: a Representative images of native PAGE analysis of proteasome complexes. Native extracts (5 μg for α subunits and 15 μg for β1, β1i, β2, β2i, β5, β5i, Rpt4, and PA28β subunits) were separated on a 4% (w/v) acrylamide gel run under native conditions in the presence of ATP and subjected to western immunoblotting using specific antibodies against the different subunits. Immunoreactive bands were imaged with a ChemiDoc system. IB: immune blot. b Proteasome activities were assayed in cell-free extracts obtained under native conditions using the fluorogenic peptide sLLVY-AMC for chymotrypsin-like activity and ANW-AMC for the β5i-associated activity of the immunoproteasome. The activity values are expressed as UF/min/µg protein and are reported in the graphs as the fold change vs day 7. (UF, Units of Fluorescence). Data are the mean ± S.D. ** p < 0.01; *** p < 0.005. The image in the right panel represents the “in-gel peptidase activity” using the fluorogenic substrate sLLVY-AMC. After complex separation by native PAGE, the gels were incubated with the substrate, and fluorescent signals were detected in a ChemiDoc system under UV light.

Article Snippet: The membranes were blocked and then incubated overnight (o/n) at 4 °C with the following primary antibodies (1:1000 dilution): anti-α1,2,3,5,6,7 (#PW8195, pan α) from Enzo Life Sciences; anti- PSMB6 /β1 (#A4053), anti- PSMB7 /β2 (#14771), anti- PSMB10 /β2i (#A5452), anti-19S PSMC6 /Rpt4 (#A5377), anti- PSME2 /PA28β (#A5562), anti-p16 INK4a (#A11651) from ABclonal; anti- PSMB8 /β5i (#13635), anti-p21 Waf1/Cip1 (#2355), anti-LC3B (#2775), anti-PCNA (PC10) (#2586) from Cell Signaling Technology; anti- PSMB9 /β1i (C-term) (#AP21207b) and anti- PSMB5 /β5 (# ALS17241 ) from Abcepta; anti-SQSTM-1/p62 (A302-856A-M) from Bethyl; and anti-Ubiquitin kindly provided by Prof. A.L.

Techniques: Clear Native PAGE, Acrylamide Gel Assay, Western Blot, Activity Assay, Fluorescence, Incubation

Journal: Communications Biology

Article Title: Immunoproteasome remodeling in senescing human macrophages reveals the loss of PA28αβ capping as a hallmark of immunosenescence

doi: 10.1038/s42003-025-08765-7

Figure Lengend Snippet: a Cells were stimulated with IFN-γ and harvested after 24 h. Two micrograms of each macrophage extract was separated via SDS‒PAGE on a 12% (w/v) acrylamide gel and subjected to Western immunoblotting with antibodies specific for the β5i subunit of the immunoproteasome and the PA28β subunit of the PA28αβ RP. b Native extracts collected after 24 h of IFN-γ stimulation were separated on a 4% (w/v) acrylamide gel under native conditions in the presence of ATP and subjected to western immunoblotting with specific antibodies against the immunoproteasome β5i subunit, PA28β, constitutive β5 subunit and α subunits. sLLVY-AMC and ANW-AMC hydrolysis represent “in-gel peptidase activity”, as determined using fluorogenic substrates for chymotrypsin-like and β5i-associated activities, respectively. After native PAGE, the gels were incubated with the specific substrate as indicated, and the activity was detected in a ChemiDoc system under UV light. c Aggresome (red) formation under basal and IFN-γ-stimulated conditions was detected with a ProteoStat® Aggresome Detection Kit at 7, 14 and 28 days of culture. DRAQ5 was used to visualize the nuclei (blue). Images were acquired with a confocal Leica microscope (×63 oil objective) using a standard rhodamine filter set. Scale bars 20 µm. IB: immune blot.

Article Snippet: The membranes were blocked and then incubated overnight (o/n) at 4 °C with the following primary antibodies (1:1000 dilution): anti-α1,2,3,5,6,7 (#PW8195, pan α) from Enzo Life Sciences; anti- PSMB6 /β1 (#A4053), anti- PSMB7 /β2 (#14771), anti- PSMB10 /β2i (#A5452), anti-19S PSMC6 /Rpt4 (#A5377), anti- PSME2 /PA28β (#A5562), anti-p16 INK4a (#A11651) from ABclonal; anti- PSMB8 /β5i (#13635), anti-p21 Waf1/Cip1 (#2355), anti-LC3B (#2775), anti-PCNA (PC10) (#2586) from Cell Signaling Technology; anti- PSMB9 /β1i (C-term) (#AP21207b) and anti- PSMB5 /β5 (# ALS17241 ) from Abcepta; anti-SQSTM-1/p62 (A302-856A-M) from Bethyl; and anti-Ubiquitin kindly provided by Prof. A.L.

Techniques: Acrylamide Gel Assay, Western Blot, Activity Assay, Clear Native PAGE, Incubation, Microscopy

Journal: Communications Biology

Article Title: Immunoproteasome remodeling in senescing human macrophages reveals the loss of PA28αβ capping as a hallmark of immunosenescence

doi: 10.1038/s42003-025-08765-7

Figure Lengend Snippet: Representative images of native PAGE analysis of proteasome complexes in the lymph nodes of young and old C57BL/6 ( a ) and BTBR ( b ) mice. Lymph node homogenates (20 μg) were separated on a 4% (w/v) polyacrylamide gel run under native conditions in the presence of ATP and subjected to western immunoblotting using specific antibodies against the immunoproteasome β5i, PA28β, constitutive β5 and α subunits. sLLVY-AMC and ANW-AMC hydrolysis represents “in-gel peptidase activity”, as determined using fluorogenic substrates for chymotrypsin-like activity and β5i-associated activity, respectively. After native electrophoresis, the gels were incubated in a solution containing the indicated specific substrates, and the activities were detected in a ChemiDoc system under UV light. c The graphs show the quantification of 20S/PA28αβ complexes normalized to the 20S content as determined by the ratio between the PA28β and panα antibody signals. The values obtained from old mice ( n = 5) are expressed as the fold change vs. values for young mice ( n = 5). The data sets are depicted as scatter plots where the bar corresponds to the median value. No data points were excluded. * p < 0.05; *** p < 0.005.

Article Snippet: The membranes were blocked and then incubated overnight (o/n) at 4 °C with the following primary antibodies (1:1000 dilution): anti-α1,2,3,5,6,7 (#PW8195, pan α) from Enzo Life Sciences; anti- PSMB6 /β1 (#A4053), anti- PSMB7 /β2 (#14771), anti- PSMB10 /β2i (#A5452), anti-19S PSMC6 /Rpt4 (#A5377), anti- PSME2 /PA28β (#A5562), anti-p16 INK4a (#A11651) from ABclonal; anti- PSMB8 /β5i (#13635), anti-p21 Waf1/Cip1 (#2355), anti-LC3B (#2775), anti-PCNA (PC10) (#2586) from Cell Signaling Technology; anti- PSMB9 /β1i (C-term) (#AP21207b) and anti- PSMB5 /β5 (# ALS17241 ) from Abcepta; anti-SQSTM-1/p62 (A302-856A-M) from Bethyl; and anti-Ubiquitin kindly provided by Prof. A.L.

Techniques: Clear Native PAGE, Western Blot, Activity Assay, Electrophoresis, Incubation