Journal: Cytotechnology

Article Title: An investigation of the mechanisms underlying the proteasome inhibitor bortezomib resistance in PC3 prostate cancer cell line

doi: 10.1007/s10616-019-00362-x

Figure Lengend Snippet: Western blot analysis of polyubiquitin conjugates, PSMB5 and cyclin D1 in parental and resistant PC3 cells. Cells were treated with 10 nM and 100 nM bortezomib for 24 h. Afterwards, 35 µg protein was separated on 12% SDS-PAGE followed by Western blot analysis as described in detail in “Materials and methods” section. PC3-P, parental PC3 cells; PC3-R, resistant PC3 cells. The result is representative of two experiments, each run in duplicate

Article Snippet: The membranes were then probed with anti-ubiquitin rabbit polyclonal antibody (1:200, cat. no. sc-9133, Santa Cruz Biotechnologies Inc.), anti-PSMB5 rabbit polyclonal antibody (1:750, cat. no. 12919S, Cell Signalling Technology Inc.) or cyclin D1 rabbit polyclonal antibody (1:750, cat. no. 2922S, Cell Signalling Technology Inc.) in TBS-T for 1 h. To determine equal protein loading, the membranes were also detected with an anti-β-actin rabbit polyclonal antibody (1:3000, cat. no. ab8227, Abcam) in TBS-T for 1 h. To visualize the specific protein bands, Phototope-HRP Western blot detection system was used.

Techniques: Western Blot, SDS Page

Journal: bioRxiv

Article Title: A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

doi: 10.1101/707661

Figure Lengend Snippet: (A) MCF7 WT cells were transfected with or without RNF4 and pre-treated with or without MG132, followed by CPT (1 μM, 2 h) co-treatment. RNF4-KO cells were pre-treated with or without MG132, then co-treated with CPT (1 μM, 2 h) followed by ICE assay for TOP1-DPC detection. (B) Same as panel (A) except that CPT was replaced by ETP (10 μM, 2 h) for ICE assay detecting TOP2α-DPCs. (C) Same as panel (B) except that IB was performed with TOP2β antibody. (D) HEK293 cells were transfected with empty vector, HA-Ub K48 or HA-K63, followed by transfection with RNF4 as indicated. Following CPT treatment (20 μM, 1 h), cells were subjected to DUST assay using anti-HA antibody. (E) Same as panel (D) except that cells were treated with ETP (200 μM, 1h) for HA-Ub-TOP2-DPC detection. (F) HEK293 cells transfected with control siRNA or RNF4 siRNA and PSMD14-FLAG expression plasmid were pre-treated with MG132 before co-treatment with CPT (20 μM, 1 h). Cells were then subjected to FLAG-IP and IB with anti-TOP1 and -TOP1-DPC antibodies. NT, no transfection. (G) Same as panel (F) except that cells were treated with ETP (200 μM, 1h) and that IB was performed with anti-TOP2α and β antibodies. (H) HEK293 cells transfected with control siRNA or RNF4 siRNA and PSMB5-FLAG expression plasmid were pre-treated with MG132 before co-treatment with CPT (20 μM, 1 h). Cells were then subjected to FLAG-IP and IB with anti-TOP1 and -TOP1-DPC antibodies. (I) Same as panel (H) except that cells were treated with ETP (200 μM, 1h and that IB was performed with anti-TOP2α and β antibodies.

Article Snippet: PSMB5-Myc-FLAG WT plasmid was purchased from OriGene (CAT#: RC209326L3). siRNA transfections were performed using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Expressing

Journal: iScience

Article Title: Ubiquitination and degradation of CD47 enhances macrophage phagocytosis of hemolytic erythrocytes

doi: 10.1016/j.isci.2025.114499

Figure Lengend Snippet: Changes in the major protein components of the proteasome during hemolysis (A) Western blot analysis of proteasomal subunits (PSME1/2, PSMB5/6/7) expression in control and hemolysis groups. (B and C) Quantification of proteasomal subunits (PSME1/2, PSMB5/6/7) expression in the RBC membrane (B) and cytoplasm (C) ( n = 3). (D) Representative immunofluorescence images of proteasomal subunits (PSME1/2, PSMB5/6/7) of hemolytic RBCs ( n = 6). Scale bars = 10 μm. (E) Schematic illustration of the animal model of immune hemolysis. (F and G) Immunofluorescence of proteasomal subunits (PSME1/2, PSMB5/6/7) in hemolytic mouse RBCs (F) and AIHA patient RBCs (G) ( n = 6). Scale bars = 10 μm. (H–J) Statistical analysis of caspase-like activity (H), trypsin-like activity (I), and chymotrypsin-like activity (J) of the membrane proteins after hemolysis ( n = 6). Data were analyzed by Student’s t test (two groups) or one-way ANOVA with Tukey’s test (multiple groups) and are expressed as mean ± SEM. ∗ p < 0.05, ∗∗∗ p < 0.001, ns = no significance.

Article Snippet: The primary antibodies utilized in this procedure were CD47 (Santa Cruz Biotechnology, Cat# sc-12730), MARCH1 (HUABIO Biotechnology, Cat# ER63906), PSME1 (Abcam, Cat# ab186832), PSME2 (Abcam, Cat# ab183727), PSMB5 (BOSTER, Cat# A03418-1), PSMB6 (ABclonal Technology, Cat# A4053), PSMB7 (BOSTER, Cat# A08095-1), and UBQLN1 (Proteintech Group, Cat# 22126-1-AP).

Techniques: Western Blot, Expressing, Control, Membrane, Immunofluorescence, Animal Model, Activity Assay

Journal: Cell Reports Methods

Article Title: BOMA, a machine-learning framework for comparative gene expression analysis across brains and organoids

doi: 10.1016/j.crmeth.2023.100409

Figure Lengend Snippet:

Article Snippet: Primary antibodies - anti-BRN2 (mouse, 1:500, Santa Cruz, SC-393324), anti-PSMB5 (rabbit, 1:1000, Novus Bio, NBP-13820) or anti-SATB2 (mouse,1:100, Gen Way, 20-372-60065) were diluted in blocking solution and incubated with tissue sections for 24 h at 4°C.

Techniques: Software

Journal: Autophagy

Article Title: Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models

doi: 10.1080/15548627.2021.2016256

Figure Lengend Snippet: Downregulation of the Psmb5 (proteasome (prosome, macropain) subunit, beta type 5) gene increases both the endogenous rat oligodendroglial and overexpressed human SNCA protein levels, whereas overexpressed TPPP/p25A levels remain unaltered. (A-D) The levels of ubiquitinated proteins (UBB) are increased upon Psmb5 downregulation. (A) Representative immunofluorescence images of OLN-93 cells treated with siRNAs targeting the rat Psmb5 gene (Psmb5 si, 10 nM) for 72 h. Scrambled RNA sequences (scr) were used as negative control. The antibodies utilized were against TUBA (green) and UBB (red). DAPI was used as a nuclear marker. Scale bar: 5 μm. (B) Quantification of UBB protein levels in OLN-93 cells, measured as M.F.I./cell. Data are expressed as the mean ± SE of three independent experiments with duplicate samples/condition within each experiment; *p < 0.05, by Student’s unpaired t test. (C) Representative immunoblots of RIPA-soluble protein cell extracts probed for poly-ubiquitinated (poly-UBB) proteins and ACTB (as loading control) antibodies, demonstrating the accumulation of poly-UBB proteins following Psmb5 silencing. (D) Quantification of the poly-UBB protein levels in OLN-93 cells treated with scr or Psmb5 siRNAs for 72 h. Data are expressed as the mean ± SE of three independent experiments with duplicate samples/condition within each experiment; *p < 0.05, by Student’s unpaired t test. (E) Quantification of the CT-like proteasomal activity in OLN-93 cells treated with Psmb5 or scr siRNAs for 72 h. Data are expressed as the mean ± SE of three independent experiments with duplicate samples/condition within each experiment; *p < 0.05; ***p < 0.001, by one-way ANOVA with Tukey’s post hoc test (to compare between the epox-treated and the scr- or Psmb5 siRNAs-treated cells), ###p < 0.001, by one-way ANOVA with Tukey’s post hoc test (to compare between the scr- and Psmb5 siRNAs-treated cells). (Fi) Representative immunofluorescence images of OLN-93, OLN-AS7 and OLN-p25α cells treated with scr or Psmb5 siRNAs (10 nM) for 72 h. The antibodies utilized were against TUBA (green) and rat SNCA (red, D37A6 antibody). DAPI was used as a nuclear marker. Scale bar: 25 μm. (Fii) Quantification of the endogenous rat SNCA protein levels measured as μm2 area surface/cell following treatment of OLN cells with Psmb5 or scr siRNAs for 72 h. Data are expressed as the mean ± SE of three independent experiments with duplicate samples/condition within each experiment; p**<0.01; by one-way ANOVA with Tukey’s post hoc test (to compare between siRNA-treated and untreated cells) or #p < 0.05; ##p < 0.01 by two-way ANOVA with Bonferroni’s correction (to compare between the different cell lines). (Gi) Representative immunofluorescence images of OLN-AS7 cells showing the increased protein levels of human SNCA following siRNA delivery targeting Psmb5 using antibodies against human SNCA (red, LB509 antibody) and TUBA (green) and DAPI staining. Scale bar: 25 μm. (Gii) Quantification of human SNCA protein levels in scr- or Psmb5 siRNA (10 nM, for 72 h) transfected OLN-AS7 cells measured as M.F.I./cell. Data are expressed as the mean ± SE of three independent experiments with duplicate samples/condition within each experiment; *p < 0.05; by Student’s unpaired t test. (Hi) Representative immunofluorescence images of OLN-p25α cells showing the protein levels of human TPPP/p25A upon molecular inhibition of the proteasome using antibodies against human TPPP/p25A (red) and TUBA (green) and DAPI staining. Scale bar: 25 μm. (Hii) Quantification of human TPPP/p25A protein levels in scr- or Psmb5 siRNA (10 nM, for 72 h) transfected OLN-p25α cells measured as M.F.I./cell. Data are expressed as the mean ± SE of three independent experiments with duplicate samples/condition within each experiment.

Article Snippet: In particular, OLN cells were transfected with Lsi1 and Lsi2 ( Lamp2a siRNAs, final concentration 60 nM), Atg5 siRNAs (rn.Ri.Atg5.13.1/2/3 TriFECTa DsiRNA kit, final concentration 10 nM), Psmb5 siRNAs ( Psmb5 Rat siRNA Oligo Duplex, Origene, SR512544; final concentration 10 nM) or control scrambled siRNA (MISSION siRNA Universal Negative Control 1, SIC001, MERCK, final concentration 60 nM and SR30004, Universal scrambled negative control siRNA duplex, Origene, final concentration 10 nM; SR30004) in Lipofectamine 2000 (Invitrogen, 11,668,019)-containing solution for 72 h. The siRNAs sequences targeting the rat Lamp2a and Psmb5 are shown in .

Techniques: Immunofluorescence, Negative Control, Marker, Western Blot, Control, Activity Assay, Staining, Transfection, Inhibition

Journal: Autophagy

Article Title: Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models

doi: 10.1080/15548627.2021.2016256

Figure Lengend Snippet: A table depicting the sequences of the siRNAs targeting the rat Lamp2a and the rat Psmb5 , as well as the human SNCA, TPPP/p25α and rat Gapdh primer sequences utilized in the RT-PCR.

Article Snippet: In particular, OLN cells were transfected with Lsi1 and Lsi2 ( Lamp2a siRNAs, final concentration 60 nM), Atg5 siRNAs (rn.Ri.Atg5.13.1/2/3 TriFECTa DsiRNA kit, final concentration 10 nM), Psmb5 siRNAs ( Psmb5 Rat siRNA Oligo Duplex, Origene, SR512544; final concentration 10 nM) or control scrambled siRNA (MISSION siRNA Universal Negative Control 1, SIC001, MERCK, final concentration 60 nM and SR30004, Universal scrambled negative control siRNA duplex, Origene, final concentration 10 nM; SR30004) in Lipofectamine 2000 (Invitrogen, 11,668,019)-containing solution for 72 h. The siRNAs sequences targeting the rat Lamp2a and Psmb5 are shown in .

Techniques: Sequencing