Anti-Proteasome 20S C2/HC2 抗体

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Proteasome 20S C2/HC2 antibody (AB3325)

Immunofluorescence analysis of 70% confluent log phase MDA-MB-231 (Human breast adenocarcinoma cell line) cells labeling Proteasome 20S C2/HC2 (green) with ab3325 at 2 μg/mL. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with ab3325 in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) secondary antibody, Alexa Fluor® 488 conjugate at 1/2000 dilution for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin. Panel d represents the merged image showing cytoplasmic localization. Panel e shows the control without primary antibody. The images were captured at 60X magnification.

• WB

Supplier Data

Western blot - Anti-Proteasome 20S C2/HC2 antibody (AB3325)

Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel, XCell SureLock™ Electrophoresis System and Novex® Sharp Pre-Stained Protein Standard. Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System. The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate.

All lanes:

Western blot - Anti-Proteasome 20S C2/HC2 antibody (ab3325) at 2 µg/mL

Lane 1:

MDA-MB-231 (Human breast adenocarcinoma cell line) whole cell lysate at 30 µg

Lane 2:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 30 µg

Lane 3:

PC-3 (Human prostate adenocarcinoma cell line) whole cell lysate at 30 µg

Lane 4:

HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 30 µg

Lane 5:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 30 µg

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) HRP cpnjugate at 0.4 µg/mL

Predicted band size: 30 kDa

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• WB

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Western blot - Anti-Proteasome 20S C2/HC2 antibody (AB3325)

All lanes:

Western blot - Anti-Proteasome 20S C2/HC2 antibody (ab3325) at 3 µg/mL

All lanes:

CHO (Chinese hamster ovary cell line) whole cell lysate

Predicted band size: 30 kDa

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• WB

Supplier Data

Western blot - Anti-Proteasome 20S C2/HC2 antibody (AB3325)

All lanes:

Western blot - Anti-Proteasome 20S C2/HC2 antibody (ab3325) at 2 µg/mL

Lane 1:

Untransfected Hep G2 whole cell extract.

Lane 2:

Proteasome 20S C2/HC2 non-targeting scrambled siRNA transfected Hep G2 whole cell extract.

Lane 3:

Proteasome 20S C2/HC2 knockdown Hep G2 whole cell extract.

Secondary

All lanes:

Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/4000 dilution

Predicted band size: 30 kDa

Observed band size: 45 kDa

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• WB

CiteAb

Western blot - Anti-Proteasome 20S C2/HC2 antibody (AB3325)

Western Blotting using Anti-Proteasome 20S C2/HC2 antibody, ab3325. Publication image from Kim, M. J. et al., 2023, Nat Commun, 37433777. Legend direct from paper.

Mitochondrial complex I deficiency increases PSMB9 mRNA levels and proteasome activity.a RNA-seq analysis of 20 S (left panel) and 19 S (right panel) proteasome components gene expression log2 fold changes (log2FC) in mitochondrial complex I-deficient HEK293T cells compared to WT HEK293T cells (n = 4). Up- and down-regulated genes (q-value < 0.05) are shown in green and pink, respectively. An immunoproteasome subunit is shown in blue. The intensity of the color shades depends on the level of expression change. Gray indicates genes with not statistically significant expression changes. b mRNA expression patterns of selected transcripts validated by RT-qPCR. The mRNA levels are presented as fold changes relative to WT. Data shown are mean ± SD (n = 3 biological replicates with two technical replicates). p-value from an ordinary one-way ANOVA with Dunnett’s multiple comparisons test using GraphPad Prism. c Chymotrypsin-like and caspase-like proteasome activities in cell lysates presented as fold changes relative to WT. Data shown are mean ± SD (n = 5 biological replicates with one~three technical replicates). **p < 0.01, ***p < 0.001 from an ordinary one-way ANOVA with Dunnett’s multiple comparisons test using GraphPad Prism. d Proteasome species in NDUFA11 KO, NDUFA13 KO and WT HEK293T cell extracts resolved by electrophoresis in 4.5% native gel followed by western blot analysis detecting a 20 S proteasome subunit PSMA1 and a 19 S proteasome subunit PSMD1 to characterize 26 S (RP2CP, doubly capped 26 S; RP1CP, singly capped 26 S) and 20 S (CP, core particle) proteasomes. Data shown are representative of four independent experiments. e Quantification of proteasomes in d using ImageJ. PSMA1 was used to quantify CP, PSMD1 was used to quantify RP1CP and RP2CP. The protein levels are presented as fold changes relative to WT. Data shown are mean ± SD (n = 4). p-value from an ordinary one-way ANOVA with Dunnett’s multiple comparisons test using GraphPad Prism. f Western blot analysis of proteasome subunit expression performed in whole cell lysates of mitochondrial complex I-deficient and WT HEK293T cells. ACTB was used as a loading control. Data shown are representative of three independent experiments. Source data are provided as a Source Data file.

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