vector control plasmid prk5 myc  (Addgene inc)

Journal: Cell reports

Article Title: The mTORC1/S6K/PDCD4/eIF4A Axis Determines Outcome of Mitotic Arrest.

doi: 10.1016/j.celrep.2020.108230

Figure Lengend Snippet: Figure 4. Active mTORC1 Promotes Survival in Response to Mitotic Poisons (A) HeLa cells were transfected in triplicate with either pRK5-myc raptor WT or 8A mutant and synchronized using thymidine/nocodazole protocol, then labeled with [S35] methionine/cysteine, and the specific activity of incorporation into equal amounts of protein was determined by trichloroacetic acid (TCA) precipitation and scintillation counting. Data are expressed as means ± SEMs. Comparison was calculated using unpaired two-tailed Student’s t test (*p < 0.01). (B) Immunoblot analysis of HeLa cells that were transfected and synchronized as in (A). Nocodazole-arrested cells were collected and released into fresh media. Degradation of cyclin B1 and phosphorylation of cdc27 are used as markers for exit from mitosis. (C) The length of one whole cell cycle (between two mitoses) was measured for 50 cells (transfected with EGFP raptor WT or 8A) using time-lapse microscopy. Comparison was calculated using unpaired two-tailed Student’s t test (ns, p > 0.05). (D) HeLa cells were transfected with either EGFP-empty vector or Raptor WT or 8A mutant. Twenty-four hours following transfection, cells were synchronized by RO3306. After 20 h, cells were washed twice with PBS and released into fresh media containing either 100 nM Taxol (top panel) or 100 nM Taxol + 200 nM rapamycin, and time-lapse imaging was started. The length of time spent by 100 cells from mitotic entry until death was plotted. Mean death time is shown as a point estimate ± SEM and as a bar plot (right panel) for each treatment. Comparisons were calculated using one-way ANOVA with Bonferroni’s multiple com- parison tests (*p < 0.01; ns, p > 0.05).

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Plasmid: pcDNA3-Flag mTOR wt (Vilella-Bach et al., 1999) Addgene Plasmid # #26603 Plasmid: myc-mTOR (Sarbassov et al., 2004) Addgene Plasmid # #1861 Plasmid: pRK5 Flag PRAS40 (Sancak et al., 2007) Addgene Plasmid # #14950 Plasmid: pRK5-myc-PRAS40 (Vander Haar et al., 2007) Addgene Plasmid # #15476 Plasmid: pRK5- myc-empty vector Dr.

Techniques: Transfection, Mutagenesis, Labeling, Activity Assay, TCA Precipitation, Comparison, Two Tailed Test, Western Blot, Phospho-proteomics, Time-lapse Microscopy, Plasmid Preparation, Imaging

Journal: Frontiers in Cell and Developmental Biology

Article Title: Inhibition of GSK3 Represses the Expression of Retinoic Acid Synthetic Enzyme ALDH1A2 via Wnt/β-Catenin Signaling in WiT49 Cells

doi: 10.3389/fcell.2020.00094

Figure Lengend Snippet: Wnts affect ALDH1A2 expression in WiT49 cells. (A) Effects of Wnts on TCF reporter activity in WiT49 cells. 400 ng TOPFlash or FOPFlash constructs with 100 ng pRK5-mWnts expression vectors or pRK-5 empty vector, as well as 25 ng of the pRL-TK renilla control vector, was transiently transfected into WiT49 cells for 48 h. The pRK5-mWnts include pRK5-mWnt1, pRK5-mWnt2b, pRK5-mWnt3a, pRK5-mWnt4, pRK5-mWnt5a, pRK5-mWnt6, pRK5-mWnt7a, pRK5-mWnt7b, pRK5-mWnt9b, and pRK5-mWnt11. Luciferase reporter activities relative to renilla (pRL-TK) are shown. Values are the mean ± SD of four determinations; representative results from at least three separate experiments are shown. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with their respective controls, calculated using the Student’s t -test. (B,C) Effect of Wnts on ALDH1A2 expression in WiT49 cells. WiT49 cells were transiently transfected with pRK5-mWnt1, pRK5-mWnt3a, pRK5-mWnt4, pRK5-mWnt9b, or the empty vector pRK5 for 48 h. The ALDH1A2 expression relative to TBP was measured by real-time RT-PCR (B) . Values are the mean ± SD of three determinations; representative results from at least three separate experiments. ∗∗ P < 0.01 and ∗∗∗ P < 0.001 compared with the empty vector pRK5 control group. The ALDH1A2 protein expression was measured by Western blot (C) . pRM-67-ALDH1A2, positive control of ALDH1A2 protein made by transfection of mammalian expression vector pReceiver-M67-ALDH1A2 to WiT49 cells. pRM-67, negative control of ALDH1A2 protein generated by transfection of pReceiver-M67 empty vector. Per lane, 50 μg of the sample was loaded, and 2 μg ALDH1A2 protein-positive control was loaded. Mock represents samples of cells treated with transfection reagent and without plasmid DNA. Values under bands were calculated by the density of bands of ALDH1A2 divided by the density of bands of GAPDH.

Article Snippet: Expression plasmids of multiple Wnts, including pRK5-mWnt1 (Addgene #42273), pRK5-mWnt2b (Addgene #42275), pRK5-mWnt3a (Addgene #42277), pRK5-mWnt4 (Addgene #42278), pRK5-mWnt5a (Addgene #42279), pRK5-mWnt6 (Addgene #42281), pRK5-mWnt7a (Addgene #42282), pRK5-mWnt7b (Addgene #42283), pRK5-mWnt9b (Addgene #42287) and pRK5-mWnt11 (Addgene #42290), were gifts from Chris Garcia and Jeremy Nathans ( ). pRK5 empty vector was made by re-ligation of the backbone of pRK5-mWnt4 (Addgene #42278) digested by Pst I. ALDH1A2 promoter or intron1G was cloned using the primers shown in .

Techniques: Expressing, Activity Assay, Construct, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Western Blot, Positive Control, Negative Control, Generated

Journal: Journal of neurochemistry

Article Title: Degradation of gamma secretase activating protein by the ubiquitin-proteasome pathway

doi: 10.1111/jnc.13011

Figure Lengend Snippet: A. N2A-APPswe were cells transfected with empty vector or GSAP plasmid, then treated with the selective E3 UB ligase inhibitor SMER3 (15μM), the pan reversible inhibitor of deubiquitinases (DUBs) PR-619(10μM), and the proteasome inhibitors Z-IE (5μM) overnight. Upper panel. Cell lysates were immunoprecipitated (IP) with anti-GSAP antibody to pull down GSAP-associated proteins, the precipitates were then assayed by immunoblot analysis (IB) with anti-ubiquitin antibody to detect all the ubiquitinated GSAP. Lower panel. Total cell lysates from the condition described in A are directly assayed by Western blot (WB) with an antibody against ubiquitin. B. N2A-APPswe cells were transiently co-transfected with GSAP plus empty vector pRK5, or HA-tagged ubiquitin wilde type (WT), or HA-tagged ubiquitin mutant with only lysine 48 and mutation of all other lysines to arginines (K48), or HA-tagged ubiquitin mutant in which all lysines were mutated to arginines (KO). Upper panel. Cell lysates were first immunoprecipitated (IP) with anti-GSAP antibody to pull down GSAP-associated proteins, then the resultant immunoprecipated product was immunoblotted (IB) with an anti-HA antibody to detect all the ubiquitin in GSAP-containing immunoprecipitates. Lower panel. Total cell lysates from the condition described in B that are directly analyzed by Western Blot (WB) with anti-HA antibody and anti-GSAP antibody.

Article Snippet: For transfection, cells were grown to 70% confluence and separately transfected with 1 μg of empty vector pcDNA3.1 (Invitrogen, Carlsbad, CA), or GSAP cDNA (Origene, Rockville, MD), or empty vector pRK5 (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-WT (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-K48 (Addgene, Cambridge MA), or pRK5-HA-Ubiquitin-KO (Addgene, Cambridge MA), by using Lipofectamine® 2000 Transfection Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions and as previously described ( 5 , 7 ).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis