Renal extracts from whole-kidney homogenates and NRK cells were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer containing 1mM phenylmethylsulfonyl fluoride (PMSF), 1.2 mM Na3VO4, 2.5 mM NaF, and 1 mM DTT (Sigma-Aldrich, USA) and protease inhibitor cocktail (Pierce, USA). 11 (link) After lysis, the extracts were centrifuged (16000 g for 20 min at 4 °C), and the supernatant was saved as the NRK cell extract. Protein concentrations were determined with the BCA Protein Assay kit (Pierce, USA). Renal extracts (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with antibodies to β5 subunit (1:1000; Abcam, #ab3330), α3 subunit (1:1000; Abcam, #ab119419), or β-actin (loading control, 1:1000; Sigma-Aldrich, #A5441). Probed membranes were washed three times, incubated with horseradish peroxidase-conjugated secondary antibodies (1:30,000; Seracare KPL), and assayed for enhanced chemiluminescence (Thermo Fisher Scientific, USA). Densitometry was performed with AlphaEase FC software (Alpha Innotech, USA).
Ab3330
Manufactured by Abcam
Sourced in United Kingdom
Ab3330 is an Alexa Fluor® 555 conjugated antibody that can be used for immunofluorescence applications. It is a mouse monoclonal antibody that recognizes an epitope on the HA tag.
Automatically generated - may contain errors
Lab products found in correlation
Ab119419, by Merck Group (2 mentions)
A5441, by Merck Group (2 mentions)
Alphaeasefc software, by Bio-Techne (2 mentions)
Ab166628, by Abcam (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Ab180606, by Abcam (1 mentions)
Sc 25778, by Santa Cruz Biotechnology (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Ab109199, by Abcam (1 mentions)
Ab3328, by Abcam (1 mentions)
Ab47594, by Abcam (1 mentions)
Ab140601, by Abcam (1 mentions)
Ab3329, by Abcam (1 mentions)
Ab3325, by Abcam (1 mentions)
Ab134175, by Abcam (1 mentions)
Phospho eif2α s51, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
5 protocols using ab3330
1
Renal Protein Extraction and Western Blotting
2
Renal Protein Extraction and Western Blotting
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Renal extracts from whole-kidney homogenates and NRK cells were prepared with radioimmunoprecipitation assay (RIPA) lysis buffer containing 1mM phenylmethylsulfonyl fluoride (PMSF), 1.2 mM Na3VO4, 2.5 mM NaF, and 1 mM DTT (Sigma-Aldrich, USA) and protease inhibitor cocktail (Pierce, USA). 11 (link) After lysis, the extracts were centrifuged (16000 g for 20 min at 4 °C), and the supernatant was saved as the NRK cell extract. Protein concentrations were determined with the BCA Protein Assay kit (Pierce, USA). Renal extracts (20 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membranes were incubated with antibodies to β5 subunit (1:1000; Abcam, #ab3330), α3 subunit (1:1000; Abcam, #ab119419), or β-actin (loading control, 1:1000; Sigma-Aldrich, #A5441). Probed membranes were washed three times, incubated with horseradish peroxidase-conjugated secondary antibodies (1:30,000; Seracare KPL), and assayed for enhanced chemiluminescence (Thermo Fisher Scientific, USA). Densitometry was performed with AlphaEase FC software (Alpha Innotech, USA).
3
Protein Expression Analysis of BI 6727 Treated A375 Cells
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5 × 105 A375
cells were plated and grown in a 10 cm culture dish and treated with
BI 6727 at 25 nM, 100 nM, or vehicle control, as previously described.
Following 24 h treatment, cells were trypsinized, washed with ice-cold
PBS, and lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40,
0.5% deoxycholic acid, 0.1% SDS) with phenylmethylsulfonyl fluoride
(PMSF) and protease inhibitor cocktail (Pierce, IL). Protein concentration
was measured with BCA Protein Assay (Pierce, IL). For immunoblot analysis,
30 μg of protein was subjected to sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) using Mini-PROTEAN TGX precast gels
and transferred onto nitrocellulose membrane. Blots were blocked in
5% nonfat dry milk in tris-buffered saline +0.1% tween-20 (TBST),
followed by probing with desired primary antibodies: anti-LDHA, AurkB,
p53, β-actin (Cell Signaling nos. 2012, 3094, 9282, 4970), PSMB1,
PSMB2, or PSMB5 (Abcam nos. ab135830, ab166628, ab3330). Blots were
then incubated with the appropriate HRP-conjugated antibodies followed
by chemiluminescent detection (Pierce, IL).
cells were plated and grown in a 10 cm culture dish and treated with
BI 6727 at 25 nM, 100 nM, or vehicle control, as previously described.
Following 24 h treatment, cells were trypsinized, washed with ice-cold
PBS, and lysed with RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP-40,
0.5% deoxycholic acid, 0.1% SDS) with phenylmethylsulfonyl fluoride
(PMSF) and protease inhibitor cocktail (Pierce, IL). Protein concentration
was measured with BCA Protein Assay (Pierce, IL). For immunoblot analysis,
30 μg of protein was subjected to sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE) using Mini-PROTEAN TGX precast gels
and transferred onto nitrocellulose membrane. Blots were blocked in
5% nonfat dry milk in tris-buffered saline +0.1% tween-20 (TBST),
followed by probing with desired primary antibodies: anti-LDHA, AurkB,
p53, β-actin (Cell Signaling nos. 2012, 3094, 9282, 4970), PSMB1,
PSMB2, or PSMB5 (Abcam nos. ab135830, ab166628, ab3330). Blots were
then incubated with the appropriate HRP-conjugated antibodies followed
by chemiluminescent detection (Pierce, IL).
4
Proteasome Subunits Expression Analysis
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Protein extracts (25 μg per muscle) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 12% and transferred to a polyvinylidene fluoride (PVDF) membrane, in which the remaining binding sites were blocked with 5% skim milk in tris buffer saline (TBS) pH 7.4. Then, membranes were incubated overnight with the following antibodies: anti-β5 (1:2000, Ab3330, Abcam, Cambridge, England); anti-β1 (1:1000, sc-100455, Santa Cruz Biotechnology, Dallas, TX, USA); anti-β5i (1:5000, Ab180606, Abcam, Cambridge, England); anti-β1i (1:300, sc-373689, Santa Cruz Biotechnology, Dallas, TX, USA); and anti-GAPDH (1:1000, sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA). Detection was accomplished with anti-IgG antibodies coupled with horseradish peroxidase antibodies (1:1000; Dako, Denmark) and visualized by enhanced electrochemiluminescence Pierce ECL Western Blotting Substrate (Thermo Scientific, USA). Quantification on Western blot image was performed with the integrated density function of ImageJ (Bethesda, MD, USA) [21] . Proteasome subunits expression was normalized with CO animals, considered as 100% expression.
5
Protein Expression Analysis in Whole-Cell Lysates
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Whole-cell lysates were subjected to western blotting to analyze the expression of various proteins using the specific antibodies that follow. Antibodies for PSMA1 (ab3325), PSMB5 (ab3330), PSMB8 (ab3329), PSMB9 (ab3328), ubiquitinated protein (ab140601), p21 (ab109199), cyclin D (ab134175), CDK1 (phospho Y15) (ab47594), and IRE1 (phospho S724) (ab48187) were purchased from Abcam. Anti-actin antibody (#A2066) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for cyclin A (sc-271682) and cyclin B1 (sc-166210) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies for cleaved caspase 3 (#9661), PARP (#9532), phospho-histone H3 (Ser10) (#3377), CHOP (#2895), LC3 (#12741), and phospho-eIF2α (S51) (#9721) were purchased from Cell Signaling Technology (Danvers, MA, USA).
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