Samples from each age point were subjected to SDS-PAGE on 8–15% polyacrylamide gels, depending on the protein being detected. For SUMO blots, gradient gels (4–20%) were used. Separated proteins were transferred to nitrocellulose membrane and immunoblotted. The primary antibodies used were: β-III-tubulin (1:5000, T8660), β-actin (1:2500, A5441), Syntaxin1A (1:5000, S0664) all from Sigma-Aldrich. PSD95 (1:1000, AB1596), GluA1 (1:1000, AB2263), NMADR1 (1:1000, AB9864) from Millipore. Aos1 (1:200, sc-46766), Uba2 (1:200, sc-376305), SUMO-1 (1:500, sc-5308) from Santa Cruz Biotechnology. Ubc9 (1:250, 610749) from BD Biosciences; PIAS1 (1:2000, 77231) from ABCAM; PIAS3 (1:1000, AP1245a) from Abgent; SENP3 (1:4000, D20A10) Cell Signaling Technology; SUMO-2/3 (1:100, Clone 8A2, produced in house) from DRHB. Luminescent or fluorescent signal was captured with a Li-COR Odyssey Fc system and quantified using Image Studio 2.1 provided by Li-COR. For SUMO quantification the whole lane was selected and quantified as well as individual bands with strong signal.
Pias1
Manufactured by Abcam
Sourced in United States
PIAS1 is a protein-coding gene that produces the protein PIAS1. PIAS1 is involved in the regulation of various cellular processes, including transcription and signal transduction. The PIAS1 protein acts as a small ubiquitin-like modifier (SUMO) E3 ligase, which facilitates the conjugation of SUMO to target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sumo 1, by Santa Cruz Biotechnology (2 mentions)
βiii tubulin, by Merck Group (1 mentions)
Psd 95, by Merck Group (1 mentions)
Odyssey fc system, by LI COR (1 mentions)
Senp3, by Cell Signaling Technology (1 mentions)
Syntaxin1a, by Merck Group (1 mentions)
S0664, by Merck Group (1 mentions)
Ab9864, by Merck Group (1 mentions)
Image studio 2, by LI COR (1 mentions)
Glua1, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
P stat5, by Abcam (1 mentions)
Hrp conjugated affinipure goat anti mouse igg, by Proteintech (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg, by Proteintech (1 mentions)
Uvp chen studio, by Analytik Jena (1 mentions)
Stat5, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Western blot apparatus, by Bio-Rad (1 mentions)
Sumo2 3, by Abcam (1 mentions)
Sumo2 3, by Santa Cruz Biotechnology (1 mentions)
4 protocols using pias1
1
Quantitative Western Blot Analysis of SUMOylation Pathway
2
Western Blot Analysis of Colonic Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The normalized supernatants (5 μg/μL) of colonic tissues were prepared as described in Enzyme-linked Immunosorbent Assay. An equivalent amount of protein in each sample was fractionated onto sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF) with a Bio-Rad Western blot apparatus. The PVDF membranes were blocked with 5% fat-free milk or 5% bovine serum albumin and then incubated overnight with the following primary antibodies at 4°C. The primary antibodies were JAK1 (1:2000), STAT5 (1:1000), p-STAT5 (1:800), PIAS1 (1:1000), and GAPDH (1:5000) (Abcam, MA, USA). These membranes were treated with the corresponding secondary antibody [HRP-conjugated AffiniPure Goat Anti-Rabbit IgG or HRP-conjugated AffiniPure Goat Anti-Mouse IgG (1:5000–1:10000) (Proteintech, IL, United States] for 1-2 h at room temperature. Subsequently, these membranes were visualized with ECL western blot substrate. The specific protein bands were scanned with a UVP Chen Studio (Analytik Jena, Germany) and quantified using Image-Pro Plus 6.0 software (Media Cybernetic, MD, United States).
3
TFAP2A Regulation by SUMOylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was isolated 96 hours after siRNA transfection and 72-96 hours after SUMO inhibitor treatment using RIPA buffer with Halt Protease Inhibitor Cocktail (100X) (ThermoFisher Scientific, Rockford, IL, USA). Antibodies for TFAP2A (AbCam, Cambridge, MA, USA), PIAS1 (AbCam), CD44 (RD Systems, Minneapolis, MN, USA), Sumo 1 (AbCam), Sumo 2/3 (AbCam) and GAPDH (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were used for Western blot analysis. Immunoprecipitations were performed using the Pierce Co-Immunoprecipitation Kit (Thermo Fisher) using antibodies for TFAP2A (AbCam) or Sumo 1 (AbCam) and Sumo 2/3 (AbCam) with rabbit IgG as a control.
4
Immunohistochemistry of PCNA, PIAS1, and SMA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following deparaffinization through a series of graded alcohols, endogenous peroxidase activity was quenched in 3% H2O2 (in methanol; Carl Roth GmbH + Co. KG, Karlsruhe, Germany). Unspecific antigen-binding sites were blocked using 10% normal serum (in PBS; Abcam, Cambridge, UK), endogenous biotin was inhibited using the Avidin/Biotin Blocking kit (Vector Laboratories, Burlingame, CA, USA) followed by heat-induced epitope retrieval (0.01 M citrate buffer, pH 6.0; 800 W for 6 min). Sections were incubated overnight at 4 °C with antibodies directed against proliferating cell nuclear antigen (PCNA; Abcam), protein inhibitor of activated STAT1 (PIAS1; Abcam) or smooth muscle α-actin (SMA; Sigma). The next day, sections were incubated with secondary antibody (Molecular Probes Inc., Eugene, OR, USA), preformed avidin–biotin complexes (Vector Laboratories) and the peroxidase substrate 3-amino-9-ethylcarbazole (for PIAS and SMA) or 3, 3 -diaminobenzidine (for PCNA) (both Vector Laboratories) until color development. Sections were briefly counterstained with Gill’s hematoxyline (Sigma), mounted in ImmuMount (ThermoScientific, Waltham, MA, USA) and photographed (Olympus BX51 microscope, Olympus Europa SE & Co. KG, Hamburg, Germany).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!