Isolation of proteins, SDS-PAGE and immunoblotting were performed in triplicates as described previously [39 (link)]. Following antibodies were used for Western blot detecting uPAR (antibody specifically targets IID7 of uPAR and detects all uPAR isoforms containing the domain II (DII) [40 (link)]), uPA (ab24121) can detect (depending on the cell line) the immature pro-uPA (at 50 kDa) and the mature uPA (at 34 kDa), PAI-1 (ab31280, Abcam, Cambridge, MA, USA), vimentin (3390), progesterone receptor (PR, 3157), RHOC (3430, Cell Signaling Technology, Beverly, MA, USA), estrogen receptor (ER, sc-8002, Santa Cruz Biotechnology, Heidelberg, DE), HER2 (A0485, Dako, Glostrup, DK), and actin (A5441, Sigma, St. Louis, MO, USA) as loading control in BT549, MDA-MB-231, MDA-MB-361, SKBR3, T47D and MCF7 breast cancer cells. uPAR was also detected in the cell supernatants whereas all the other proteins were detected in the cell lysates.
Ab24121
Manufactured by Abcam
Sourced in United States
Ab24121 is a laboratory equipment product from Abcam. It is designed for use in scientific research applications. The core function of this product is to [PRODUCT_CORE_FUNCTION].
Automatically generated - may contain errors
Lab products found in correlation
Er sc 8002, by Santa Cruz Biotechnology (1 mentions)
Ab31280, by Abcam (1 mentions)
Her2 a0485, by Agilent Technologies (1 mentions)
Anti rabbit igg hrp 7074, by Cell Signaling Technology (1 mentions)
Clarity ecl blotting substrate, by Bio-Rad (1 mentions)
Ab227069, by Abcam (1 mentions)
Chemidoc imager, by Bio-Rad (1 mentions)
Ab47742, by Abcam (1 mentions)
Anti mouse igg hrp 7076s, by Cell Signaling Technology (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Diabzi compound 3, by Selleck Chemicals (1 mentions)
Fetal bovine serum (fbs), by ExCell Bio (1 mentions)
Gsk2606414, by Selleck Chemicals (1 mentions)
Gsk2656157, by Selleck Chemicals (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Ripa lysis kit, by Beyotime (1 mentions)
Ab209845, by Abcam (1 mentions)
6 protocols using ab24121
1
Comprehensive Protein Analysis in Breast Cancer
2
Western Blot Analysis of Angiogenic Factors
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Equal concentration protein from cell lysates and CM were separated by 12% SDS/PAGE gels. The proteins were transferred to nitrocellulose or PVDF membranes and blocked with 5% skim milk. The membranes were probed using anti‐human ALDH1A3 (OTI4E8; OriGene Technologies, Rockville, MD, USA), tPA (ab227069; Abcam, Toronto, Canada), uPA (ab24121; Abcam), and PAI‐2 (ab47742; Abcam) antibodies. Anti‐rabbit IgG‐HRP (7074S; Cell Signaling Technology) secondary antibody was used for tPA, uPA, and PAI‐2 while anti‐mouse IgG‐HRP (7076S; Cell Signaling Technology, Danvers, MA, USA) secondary antibody was used for ALDH1A3. Immuno‐reactive proteins were detected by chemiluminescence (using Clarity ECL blotting substrate (Bio‐Rad)) and visualized with images captured with a ChemiDoc Imager (Bio‐Rad). Total protein was used as a loading control.
3
STING and PERK Signaling Pathways
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MDA-MB-231, MCF7, ZR-75-1, and HFF cell lines were purchased from the ATCC. ZR-75-1 cells were cultured in RPMI 1640 (Gibco), and the other cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Excell Bio) in an incubator at 37 °C with 5% CO2. The antibody against GAPDH (sc-47724) was purchased from Senta Cruz Biotechnology. Antibodies against STING (13647), TBK1 (3504s), pTBK1 S172 (5483S), elF2α (5324S), and p-elF2α S51 (3398S) were purchased from Cell Signaling Technology. Antibodies against PLAU (ab24121) and pIRF3 S386 (ab76493) were purchased from Abcam (MA 02453, USA). Antibodies against PERK (A18196) and p-PERK T982 (AP0086) were purchased from ABclonal Technology. STING agonist diABZI (Compound 3) and PERK inhibitors GSK2656157 and GSK2606414 were purchased from Selleck Chemicals.
4
Western Blot Analysis of Protein Expression
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Total protein was extracted using a RIPA Lysis Kit (Beyotime Biotechnology, Shanghai, China) by following the manufacturer’s protocol. Equal amounts of protein from each sample were electrophoretically separated using polyacrylamide gels, and then transferred to polyvinylidene fluoride membranes. The membranes were blocked at 23–25 °C for 1 h and then incubated at 4 °C overnight with primary antibodies against GLIPR1 (ab198215, Abcam), PLAU (ab28230 and ab24121, Abcam), EGFR (ab52894, Abcam), p-EGFR (11862S, Cell Signaling Technology, Boston, MA, USA), caspase-1 (22915-1-AP, Proteintech Group, Chicago, IL, USA), GSDMD (ab209845, Abcam; 97558, Cell Signaling Technology), IL-1β (AF5103, Affinity Biosciences, Cincinnati, OH, USA), and β-actin (4970, Cell Signaling Technology). Then, the membranes were washed three times and incubated with an appropriate horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The proteins were visualized using enhanced electro-chemiluminescence reagents (Beyotime Biotechnology), and the bands were analyzed using an imaging system (Bio-Rad, Hercules, CA, USA).
5
Protein Expression Analysis via Western Blot
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Total cell lysates were prepared using the radioimmunoprecipitation assay buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholic acid, and 0.1% sodium dodecyl sulphate). Total protein was separated through 6–10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose filter membranes (Millipore, Billerica, USA). Subsequently, the membranes were blocked with a blocking buffer for 1 h at RT and incubated with the following primary antibodies: NLN (ab119802, Abcam), CCND1 (RM-9104-S, Thermo Scientific), PLAU (ab24121, Abcam) , SEPN1(sc-365824, Santa Cruz) , Actin (MAB1501, Millipore) and GAPDH (GTX627408, GeneTex) for overnight at 4 °C. Secondary labelling was performed using suitable horseradish peroxidase-conjugated secondary antibody for 1 h at RT. Finally, proteins were visualised using WesternBrightTM enhanced chemiluminescence (Advansta Inc., USA) and detected using the BioSpectrumTW 500 Imaging System (UVP, USA).
6
Western Blot Analysis of Protein Signaling
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Cells were lysed in 2× Laemmli loading buffer and denatured at 95 °C for 5 min. Then proteins were separated on 10% SDS-PAGE gel and transferred onto PVDF membranes. The membranes were blocked in 5% BSA or non-fat dried milk (NFDM) in TBS with 0.1% Tween20 (TBST) at RT for 1 h, followed by incubation at 4 °C overnight with primary antibodies: STAT3 (CST, 9139 s, 1:1000), phospho-STAT3 (CST, 9145 s, 1:1000), ERK (CST, #4695, 1:2000), phospho-ERK-Thr202/Tyr204 (CST, #9101, 1:2000), AKT (CST, 4685 s, 1:2000), phospho-AKT-S473 (CST, 4060 s, 1:2000), NRP1 (Abcam, ab81321, 1:1000), PLAU (Abcam, ab24121, 1:1000), LRP1 (Abcam, ab92544, 1:1000), and β-actin (Beyotime, AF0003, 1:5000). After washed with TBST for three times, membranes were incubated with HRP-conjugated goat anti-mouse IgG (Beyotime, A0216, 1:1000) or goat anti-rabbit IgG (Beyotime, A0208, 1:1000) at RT for 1 h. After washing three times with TBST, blots were detected with Clarity Western ECL Substrate (Bio-Rad) through a Gel Imaging system (Tanon 6100 C) or an Odyssey infrared scanner (LICOR Bioscience).
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