Plasmid px330 was originally obtained from Addgene (Cat #42230) and the SpCas9 cassette was cloned into a pcDNA3β-Hyg-based expression vector. Plasmids expressing nCas9 (D10A and H840A) were generated by site-directed mutation from pcDNA3β-Cas9 using KOD-Plus-Neo Kit (TOYOBO). Plasmids of sgRNAs were constructed from the U6-sgRNA vector as described previously46 (link). The sgRNA target sequences are listed in Supplementary Table 1 . The newly constructed plasmids were confirmed by Sanger sequencing. Chemical treatments were performed with Aphidicolin (CAS 38966-21-1, Sigma) at 5 μg/mL, L-Mimosine (S7446, Selleck) and Hydroxyurea (S1896, Selleck) at 2 mM, Bleomycin (S1214, Selleck) at 20 μg/mL, Olaparib (S1060, Selleck) at 2 μM and Camptothecin (S1288, Selleck) at 1 μM.
L mimosine
Manufactured by Selleck Chemicals
L-mimosine is a naturally occurring amino acid that can be used as a laboratory reagent. It has a chemical formula of C₆H₁₁N₃O₃ and a molecular weight of 201.17 g/mol. L-mimosine serves as a precursor in the synthesis of various chemical compounds and can be utilized in a variety of research applications.
Automatically generated - may contain errors
Lab products found in correlation
Dabrafenib, by Selleck Chemicals (2 mentions)
Vemurafenib, by Selleck Chemicals (2 mentions)
Sch772984, by Topscience (2 mentions)
Kod plus neo kit, by Toyobo (1 mentions)
Bleomycin, by Selleck Chemicals (1 mentions)
Camptothecin, by Selleck Chemicals (1 mentions)
Hydroxyurea, by Selleck Chemicals (1 mentions)
Olaparib, by Selleck Chemicals (1 mentions)
β action, by Proteintech (1 mentions)
β tubulin, by Proteintech (1 mentions)
Cck 8, by Selleck Chemicals (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Ppp2r1b, by Abcam (1 mentions)
P erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Ppp2r1b plasmid, by Gene Tech (1 mentions)
3 protocols using l mimosine
1
Plasmid Construction and Chemical Treatments
2
Investigating MAPK Signaling Modulators
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Vemurafenib, dabrafenib and L-mimosine were purchased from Selleck (Shanghai, China). The ERK inhibitor SCH772984 was purchased from TOPSCIENCE (Shanghai, China). The anti-eIF3a antibody (cat. no. ab128996, 1:1,000) and anti-PPP2R1B antibody (cat. no. ab154815, 1:1,000) were purchased from Abcam. The anti-ERK antibody (cat. no. 4695, 1:1,000) and anti-p-ERK antibody (Thr202/Tyr204) (cat. no. 4370, 1:1,000) were purchased from Cell Signaling Technologies. The anti-puromycin antibody (cat. no. EQ0001, 1:1,000) was purchased from Kerafast. The anti-Flag antibody (cat. no. M185, 1:1,000) and anti-HA antibody (cat. no. M180, 1:1,000) were purchased from MBL. β-action (1:10,000) and β-Tubulin (1:10,000) antibodies were purchased from Proteintech. An enhanced chemiluminescence (ECL) kit was purchased from Cytiva. CCK8 was purchased from Bimake (Shanghai, China).
3
Modulation of ERK signaling by eIF3a and PPP2R1B
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Vemurafenib, dabrafenib and L-mimosine were purchased from Selleck (Shanghai, China). ERK inhibitor SCH772984 was purchased from TOPSCIENCE (Shanghai, China). Antibodies used in immunoblotting: eIF3a and PPP2R1B were purchased from Abcam. ERK and p-ERK (Thr202/Tyr204) were purchased from Cell Signaling Technologies. Anti-Flag and anti-HA was purchased from MBL. β-action and β-Tubulin was purchased from Proteintech. siRNA and plasmid transfection siRNA targeting eIF3a and PPP2R1B was purchased from RiboBio (Guangzhou, China). PPP2R1B plasmid was purchased from Gene (Shanghai, China). Transfection of siRNA was carried out according to the manufacturer's protocol. Brie y, cells in exponential phase of growth were plated in six-well tissue culture plates and then transfected with siRNA using lipofectamine RNAimax (Invitrogen) reagent and OPTI-MEM medium. Transfection of the plasmid was carried out using lipofectamine 2000 (Invitrogen) reagent according to the manufacturer's protocol.
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