Establishing Cell Lines and Transfection Procedures

HL60, U937, K562, 32Dcl3 and HEK293T cells obtained from ATCC were cultured as previously described [13 (link), 23 (link), 29 (link), 42 (link)]. siMNT (Smartpool: ON-TARGET plus MNT SiRNA L-009373-00-0010), scrambled siRNA and siRNA transfection reagent DhermaFECT (ThermoScientific T-2001-03) were purchased from Dhermacon RNA technologies (Lafayette, CO, USA). shMNT (TRCN00000234786), shE6AP (TRCN0000235499) and shRNA control were purchased from Sigma. Expression plasmids for HA-MNT [43 (link)], pcDNA3.1-E6AP [10 (link)], pCAG-HA-E6AP-C843A [44 (link)], pGEX4T-GST-E6AP [45 (link)] were kind gifts from G. Meroni, Nihar Jana, Ikuo Shoji and Zafar Nawaz, respectively. Transfections in adherent cells were performed using Lipofectamine-LTX and Plus Reagent (15338-100; Invitrogen) as per manufacture`s protocol as previously described [13 (link), 42 (link)]; Transfections in myeloid cells HL60 and U937 were performed using AMAXA cell Nucleofector kit V (VCA-1003; Lonza). E6AP-C843A-HA is a catalytically inactive mutant of E6AP [13 (link)]. Cells were treated with 10μM MG132 (Z-Leu-Leu-Leu-al, Sigma, St.Louis, MO) and 10μM Lactacystin (Sigma) 6h prior to cell lysis.

Free full text: Click here

Kapoor I., Kanaujiya J., Kumar Y., Thota J.R., Bhatt M.L., Chattopadhyay N., Sanyal S, & Trivedi A.K. (2015). Proteomic discovery of MNT as a novel interacting partner of E3 ubiquitin ligase E6AP and a key mediator of myeloid differentiation. Oncotarget, 7(7), 7640-7656.

Publication 2015

shMNT shE6AP Lipofectamine-LTX Plus Reagent AMAXA cell Nucleofector kit V (Z-Leu-Leu-Leu-al Lactacystin

Cell Gifts Hl60 cells Lactacystin Lipofectamine Mg132 Myeloid cells Plasmids Shrna Sirna Transfections Z leu leu leu al

Corresponding Organization :

Other organizations : Central Drug Research Institute, King George's Medical University

Top 5 similar protocols

Variable analysis

independent variables

SiMNT (Smartpool: ON-TARGET plus MNT SiRNA L-009373-00-0010)
Scrambled siRNA
HA-MNT expression plasmid
PcDNA3.1-E6AP expression plasmid
PCAG-HA-E6AP-C843A expression plasmid
PGEX4T-GST-E6AP expression plasmid

dependent variables

Cell behavior and characteristics of HL60, U937, K562, 32Dcl3, and HEK293T cells

control variables

HL60, U937, K562, 32Dcl3, and HEK293T cells cultured as previously described in references [13], [23], [29], and [42]
SiRNA transfection reagent DhermaFECT (ThermoScientific T-2001-03)
ShRNA control
E6AP-C843A-HA (catalytically inactive mutant of E6AP)
MG132 (Z-Leu-Leu-Leu-al, Sigma, St.Louis, MO)
Lactacystin (Sigma)

controls

HA-MNT expression plasmid, pcDNA3.1-E6AP expression plasmid, pCAG-HA-E6AP-C843A expression plasmid, pGEX4T-GST-E6AP expression plasmid
Scrambled siRNA, shRNA control

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

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?

Revolutionizing how scientists
search and build protocols!