Journal: Pathogens

Article Title: Trypanosoma brucei Acyl-Protein Thioesterase-like (TbAPT-L) Is a Lipase with Esterase Activity for Short and Medium-Chain Fatty Acids but Has No Depalmitoylation Activity

doi: 10.3390/pathogens11111245

Figure Lengend Snippet: TbAPT-L is not essential for growth in vitro. ( A ) Cumulative growth curve (n = 2) for 10 days with T. brucei SM427 bloodstream form (BF) cells lines: wild type (WT), TbAPT-L pZJM (KD), and TbAPT-L plew79-2×myc (OE), with or without 1 μg/mL tetracycline (-T or +T). RNA and whole cell lysate were prepared at days 1 and 10 for analysis. ( B ) qRT-PCR analysis of APT-L expression at days 1 and 10 normalized to the housekeeping gene telomerase reverse transcriptase (TERT) as a control. Relative quantification values are noted above each bar. ( C ) Western blot analysis of whole cell lysates for TbAPT-L (25 and 30 kDa) β-tubulin (50 kDa) at days 1 and 10 from all cells and conditions (see above). Note that the TbAPT-L protein runs at a smaller size (25 kDa) in cell lysates than for the fusion protein (OE lanes). Relative band intensity was calculated by densitometry normalizing TbAPT-L to β-tubulin bands and setting WT -Tet as 1.00 for both days 1 and 10 and correcting all other samples from each timepoint to that reference.

Article Snippet: TbAPT-L (30 kDa) was detected with 1:1000 mouse anti-TbAPT-L polyclonal primary Ab and 1:20,000 LI-COR goat anti-mouse IRDye 800 secondary Ab; and subsequently re-probed without stripping for detection of β-tubulin (50 kDa) with 1:1000 mouse anti-beta tubulin E7 monoclonal Ab (Developmental Studies Hybridoma Bank at the University of Iowa) and 1:20,000 LI-COR goat anti-mouse IRDye 680 secondary Ab.

Techniques: In Vitro, Quantitative RT-PCR, Expressing, Reverse Transcription, Control, Quantitative Proteomics, Western Blot

Journal: Scientific reports

Article Title: PTPRS Regulates Colorectal Cancer RAS Pathway Activity by Inactivating Erk and Preventing Its Nuclear Translocation.

doi: 10.1038/s41598-018-27584-x

Figure Lengend Snippet: Figure 2. Western blot analysis for ERK and MEK activation. The indicated CRC cell lines were cultured, cells harvested, extracts prepared and western blots performed. (a) Cells (HCT116, SW620 and KM12L4A) cultured with ISP, an inhibitor of PTPRS, or a scrambled control peptide (SC). Western blots to detect ERK, tyrosine phosphorylated ERK, MEK and phosphorylated MEK, and AKT and phosphorylated AKT are shown as indicated. The quantitations were determined by normalizing the phosphorylated protein values with the total protein; then dividing the ISP values by the SC values. (b) The indicated cells had PTPRS knocked down with siRNA to PTPRS (siPTPRS) or were treated with a scrambled siRNA control (siCtl). Western blot analysis shows PTPRS, phospho-ERK, ERK, phospho-MEK, MEK, phospho-AKT, AKT, and alpha-tubulin. Knockdown of PTPRS via siRNA shows results consistent with the ISP treatments. (c) CRISPR knockouts of PTPRS in HCT116, SW620, and KM12L4A cell lines and their CRISPR control cell lines where cell extracts were used in western blot analysis for phosphorylation of ERK and MEK. This analysis shows PTPRS, ERK, phospho-ERK, MEK, phospho-MEK, AKT, phospho-AKT, and alpha tubulin in the cell line pairs (Ctl and KO) as indicated. (d) ddPCR analysis of PTPRS expression in CRISPR KO cells for HCT116, SW620, and KM12L4A. Analysis of the ddPCR result shows a near complete knockout for HCT116 and KM12L4A; SW620 shows >85% knockout. All experiments were done in triplicate. The mean and standard deviation are shown. Two-tailed, paired t test was used to determine the statistical significance for comparison as indicated.

Article Snippet: All primary antibodies were rabbit unless specified and were sourced as follows: PTPRS (mouse Cat. No.ab55640 Abcam); PTPRS (goat AF3430 R&D Systems); alpha-Tubulin (mouse sc-8035 Santa Cruz).

Techniques: Western Blot, Activation Assay, Cell Culture, Control, Knockdown, CRISPR, Phospho-proteomics, Expressing, Knock-Out, Standard Deviation, Two Tailed Test, Comparison

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 2 | PEDV replication inhibit expression of SLA-DR in BM-DCs (A) BM-DCs were infected by Vero cell-adapted PEDV strain KB2013-p120 at 1MOI for 24, 48 and 72 hours then harvested for qPCR analysis for mRNA level of SLA-DRa and SLA-DRb. Transcript of tubulin were analyzed from the same sample to normalize total RNA input. Error bars represent variation from at least three independent experiments. Significant differences between indicated groups was marked by **P < 0.01; ***P < 0.001. (B) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24, 48 and 72 hours then harvested for western blot to evaluate SLA-DRa, SLA-DRb and PEDV-N protein level using corresponding antibodies. Normal BM-DCs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (C) BM-DCs were infected by PEDV-KB2013-p120 strain 1MOI for 24, 48 and 72 hours then stained with anti-SLA-DR antibody followed by visualization of APC labeled goat anti-mouse IgG. Then the cells were subjected to flow cytometry analysis for evaluating cell surface expression of SLA-DR. BM-DCs without PEDV infection stained with normal mouse IgG as primary antibody were included as primary antibody isotype control. Error bars represent variation of quantification of FACS data from at least three independent experiments. Significant differences between indicated groups was marked by **P < 0.01; or ns, non significant.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Expressing, Infection, Western Blot, Control, Staining, Labeling, Cytometry

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 3 | Susceptibility of BM-DCs for PEDV is not strain-specific. (A) BM-DCs were infected by PEDV-KB2013-p120 strain at 0.5, 1 and 2MOI for 48 hours then harvested for western blot to evaluate SLA-DRa, SLA-DRb and PEDV-N protein. Normal BM-DCs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (B) BM-DCs were infected by Vero cell-adapted PEDV strain KB2013-p120, virulent PEDV strain CH/hubei/2016 (GenBank accession number: KY496315.1), and a field PEDV isolate SXYL-21 for 48 hours. Next, cells were harvested for western blot using mice serum raised against recombinant PEDV-N protein and anti-SLA-DRa Mab. Normal BM-DCs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (C) BM-DCs were infected by PEDV-KB2013-p120 strain at 1 MOI for 24 hours then 10mg LPS was added to the PEDV infected BM-DCs to stimulate the cells for another 24 hours. Next, all cells were harvested for western blot to evaluate SLA-DRa, SLA-DRb and PEDV-N protein. PEDV infected BM-DCs without LPS stimulation and normal BM-DCs cells were included as controls. Tubulin was probed from the same sample to normalize the total protein load.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Infection, Western Blot, Control, Recombinant

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 5 | PAMs are susceptible for PEDV. (A) PAMs were infected by Vero cell-adapted PEDV strain KB2013-p120 for 24, 48 and 72 hours. Next, cells were harvested for western blot using mice serum raised against recombinant PEDV-N protein and anti-SLA-DRa Mab. Normal PAMs cells without PEDV infection were included as control. Tubulin was probed from the same sample to normalize the total protein load. (B) PAMs were infected by PEDV strain KB2013-p120 for 24, 48 and 72 hours. Next, cells were harvested by TRizol for qPCR analysis of SLA-DRa and b mRNA level. Normal PAMs cells without PEDV infection were included as control. Error bars represent variation from at least three independent experiments. Significant differences between indicated groups was marked by *P < 0.05; **P < 0.01. or ns, non significant.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Infection, Western Blot, Recombinant, Control

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 6 | Inhibition of SLA-DR expression in BM-DCs did not require PEDV replication. (A) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain or UV-inactivated PEDV-KB2013-p120 strain for 24 hours. Next the cells were fixed and stained with mice serum raised against recombinant PEDV-N protein and visualized by secondary antibody. (B) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain (PEDV) or UV-inactivated PEDV-KB2013-p120 strain (UV- PEDV) for 24 hours. Next, cell culture supernatants were harvested and titrated in Vero cells. Error bars represent variation from at least three independent experiments. (C) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain (PEDV) or UV-inactivated PEDV-KB2013-p120 strain (UV-PEDV) for 24 hours. Next, cells were harvested for qPCR evaluation of mRNA level of SLA-DR a and SLA-DR b. All experiment was repeated at least for three times. Significant differences between indicated groups was marked by *P < 0.05. (D) BM-DCs were inoculated with either live PEDV-KB2013-p120 strain (PEDV) or UV-inactivated PEDV-KB2013-p120 strain (UV-PEDV) for 48 hours. Next, cells were harvested for western blot using mice serum raised against recombinant PEDV-N protein, anti- SLA-DRa Mab and mice serum raised against recombinant SLA-DRb protein. Normal BM-DCs cells were included as control. Tubulin was probed from the same sample to normalize the total protein load. ns, non significant.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Inhibition, Expressing, Staining, Recombinant, Cell Culture, Western Blot, Control

Journal: Frontiers in immunology

Article Title: Porcine Epidemic Diarrhea Virus Envelope Protein Blocks SLA-DR Expression in Barrow-Derived Dendritic Cells by Inhibiting Promoters Activation.

doi: 10.3389/fimmu.2021.741425

Figure Lengend Snippet: FIGURE 8 | Ubiquitin-proteasome pathway did not involve in inhibition of SLA-DR expression. (A) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24, then followed by treatment of either DMSO or MG132 for another 24 hours. Then cells were harvested for western blot evaluating of SLA-DRa, SLA-DRb and PEDV-N protein. Normal BM-DCs cells without PEDV infection but treated with the same dose of DMSO or MG132 were included as controls. Tubulin was probed from the same sample to normalize the total protein load. (B) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24 hours. Next, cells were harvested for western blot evaluating of universal ubiquitination level and PEDV-N protein. Normal BM-DCs cells were included as control. Tubulin was probed from the same sample to normalize the total protein load. (C) BM-DCs were infected by PEDV-KB2013-p120 strain at 1MOI for 24 hours. Next, cells were lyzed using RIPA buffer supplemented with protease inhibitor cocktail and NEM for SLA-DR enrichment using anti-SLA-DR Mab-MY533. Enriched samples were subjected to western blot using ubiquitin antibody and anti-SLA-DRa chain antibody. (D) HEK-293T-SLA-DRa/b stable cells were transfected with plasmids encoding S2, M, N, E and ORF3 for 48 hours. Next, cells were harvested for western blot to evaluate expression of SLA-DRa and b. Cells transfected with empty vector were included as control. Tubulin was probed from the same sample to normalize the total protein load.

Article Snippet: Membranes were probed with mouse serum raised against PEDV-N protein (1:200 dilution in TBS), homemade mAb against SLA-DRa (Clone No.2E11D9), homemade polyclonal antibodies against SLA-DRb, anti-b-tubulin mAb (Transgene), or anti-ubiquitin mAb (Santa Cruz Biotech, Santa Cruz, CA, USA).

Techniques: Ubiquitin Proteomics, Inhibition, Expressing, Infection, Western Blot, Control, Protease Inhibitor, Transfection, Plasmid Preparation

Journal: PLoS ONE

Article Title: βPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway

doi: 10.1371/journal.pone.0230814

Figure Lengend Snippet: (A) Representative images of WT and βPix-NIKO neurons fixed at DIV4 and stained with acetylated α-tubulin (Acet-Tub) antibody (red) and tyrosinated α-tubulin (Tyr-Tub) antibody (green). The white dashed lines indicate the longest neurite of the neurons in each group and are straightened in Fig 2B. (B) The longest neurite of the WT and βPix-NIKO neurons shown in Fig 2A is straightened using ImageJ software. (C) The distribution graph shows that Acet-Tub levels are lower in the βPix-NIKO neurons throughout the length of the longest neurite compared with those in the WT neurons. The average Acet-Tub level (values in the top right corner) is 68% lower in the βPix-NIKO neurons compared with the WT neurons. (D) The distribution graph shows that Tyr-Tub levels slightly increase along the longest neurite extending from the βPix-NIKO neurons compared with that from the WT neurons. The average Tyr-Tub level (values in the top right corner) is 1.1-fold higher in the βPix-NIKO neurons compared with the WT neurons. n = 23–39 neurons per group from three independent cultures. (E) Representative blots showing Acet-Tub and Tyr-Tub levels in WT and βPix-NIKO neurons at DIV4. Compared with WT neurons, βPix-NIKO neurons show decreased levels in Acet-Tub and increased levels in Tyr-Tub. Data from at least three independent experiments. For the comparison of WT and βPix-NIKO neurons, * P < 0.05 and *** P < 0.001 for (C) and (D) by Student’s t -tests. The yellow boxes in (C) and (D) indicate P < 0.01 by Student’s t -tests.

Article Snippet: The following commercially available antibodies were used for Western blot and immunostaining analysis: monoclonal mouse antibody against acetylated α-tubulin (clone 6-11B-1, Sigma), monoclonal rabbit antibody against acetylated α-tubulin (clone D20G3, Cell Signaling), monoclonal mouse antibody against GFP (clone B-2, Santa Cruz), polyclonal rabbit antibody against MAP2 (Cell Signaling), monoclonal mouse antibody against Myc (clone 9E10, Santa Cruz), monoclonal rabbit antibody against Stathmin1 (EP1573Y, Abcam), polyclonal rabbit antibody against Stathmin1 (phospho S16, Abcam), monoclonal mouse antibody against tyrosinated α-tubulin (clone TUB-1A2, Sigma), monoclonal mouse antibody against Tau (clone Tau-5, Chemicon), monoclonal mouse antibody against α-tubulin (clone DM1A, Abcam), and monoclonal mouse antibody against β3-tubulin (clone TU-20, Chemicon).

Techniques: Staining, Software, Comparison

Journal: PLoS ONE

Article Title: βPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway

doi: 10.1371/journal.pone.0230814

Figure Lengend Snippet: (A) Representative blots showing Acet-Tub and Tyr-Tub levels in βPix-NIKO neurons transfected with GFP, GFP-βPix-a, GFP-βPix-b, or GFP-βPix-d at DIV3 and lysed at DIV4. (B) Quantification of relative Acet-Tub expression levels for the results shown in (A) as normalized to α-tubulin expression. GFP-βPix-b or GFP-βPix-d induces a significant increase in the level of Acet-Tub, with GFP-βPix-d having a greater effect than GFP-βPix-b. (C) Quantification of relative Tyr-Tub expression levels for the results shown in (A) as normalized to α-tubulin expression. There is no significant difference in Tyr-Tub levels for the expression of GFP-βPix-a, GFP-βPix-b, or GFP-βPix-d in the βPix-NIKO neurons. (D) Representative images of βPix-NIKO neurons transfected with GFP, GFP-βPix-b, or GFP-βPix-d at DIV3, fixed at DIV4, and stained with Acet-Tub antibody (red) and Tyr-Tub antibody (blue). The white dashed lines indicate the longest neurite of the neuron in each group, which is straightened in Fig 4E. (E) The longest neurite of the βPix-NIKO neurons transfected with GFP, GFP-βPix-b, or GFP-βPix-d from Fig 4D is straightened using ImageJ software. (F) The Acet-Tub levels along the longest neurite are increased by GFP-βPix-b or GFP-βPix-d expression in the βPix-NIKO neurons compared with the GFP-expressing control. (G) There is little significant change in Tyr-Tub levels along the longest neurite following the expression of GFP-βPix-b or GFP-βPix-d in the βPix-NIKO neurons. Five independent cultures for (A)–(C) and n = 61–82 neurons per group from three independent cultures for (D)–(G). For the comparison among hippocampal neurons from neuronal βPix isoform KO mice transfected with GFP, GFP-βPix-b, and GFP-βPix-d, * P < 0.05 and *** P < 0.001 for (B) by one-way ANOVA with post-hoc Tukey’s test. Pink boxes in (F) denote P < 0.01 for the comparison of GFP and GFP-βPix-b and yellow boxes in (F) and (G) denote P < 0.01 for the comparison of GFP and GFP-βPix-d by Student’s t -tests.

Article Snippet: The following commercially available antibodies were used for Western blot and immunostaining analysis: monoclonal mouse antibody against acetylated α-tubulin (clone 6-11B-1, Sigma), monoclonal rabbit antibody against acetylated α-tubulin (clone D20G3, Cell Signaling), monoclonal mouse antibody against GFP (clone B-2, Santa Cruz), polyclonal rabbit antibody against MAP2 (Cell Signaling), monoclonal mouse antibody against Myc (clone 9E10, Santa Cruz), monoclonal rabbit antibody against Stathmin1 (EP1573Y, Abcam), polyclonal rabbit antibody against Stathmin1 (phospho S16, Abcam), monoclonal mouse antibody against tyrosinated α-tubulin (clone TUB-1A2, Sigma), monoclonal mouse antibody against Tau (clone Tau-5, Chemicon), monoclonal mouse antibody against α-tubulin (clone DM1A, Abcam), and monoclonal mouse antibody against β3-tubulin (clone TU-20, Chemicon).

Techniques: Transfection, Expressing, Staining, Software, Control, Comparison

Journal: PLoS ONE

Article Title: βPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway

doi: 10.1371/journal.pone.0230814

Figure Lengend Snippet: (A) Representative images of WT neurons transfected with GFP and βPix-NIKO neurons transfected with GFP or GFP-βPix-d at DIV3, fixed at DIV4, and stained with p-Stathmin1 (S16) (red) and β3-tubulin antibodies (blue). (B) The length of the longest neurite decreases by 60% in the βPix-NIKO neurons expressing GFP (NIKO+GFP) compared with the WT neurons transfected with GFP (WT+GFP). The expression of GFP-βPix-d in the βPix-NIKO neurons rescues the defects in neurite length. (C) Total neurite length decreases by 62% in the NIKO+GFP neurons compared with the WT+GFP neurons. The expression of GFP-βPix-d in the βPix-NIKO neurons rescues defects in neurite length. (D) Total branching number decreases by 73% in the NIKO+GFP neurons compared with the WT+GFP neurons. The expression of GFP-βPix-d in the βPix-NIKO neurons rescues the defect in branch numbers. (E) The longest neurite shown in Fig 5A are straightened using ImageJ software. (F) In the longest neurite, the phosphorylation levels of Stathmin1 at Ser16, which are normalized to β3-tubulin, decreases by 20% in the NIKO+GFP neurons compared with the WT+GFP neurons. The expression of GFP-βPix-d in the βPix-NIKO neurons rescues the phosphorylation levels of Stathmin1 at Ser16. (G) In the longest neurite, the phosphorylation of Stathmin1 at Ser16 is significantly lower in the NIKO+GFP neurons compared with the WT+GFP neurons and is recovered by the expression of GFP-βPix-d in the βPix-NIKO neurons. (H) Representative blots for the phosphorylation level of Stathmin1 at Ser16 in WT neurons transfected with GFP and βPix-NIKO neurons transfected with GFP or GFP-βPix-d at DIV3 and lysed at DIV4. (I) Quantification of relative expression levels for the results shown in (H) as normalized to α-tubulin expression. Compared with the WT+GFP neurons, the NIKO+GFP neurons show a 41% decrease in the ratio of p-Stathmin1 at Ser16 to total Stathmin1, and this ratio is recovered by the expression of GFP-βPix-d in the βPix-NIKO neurons. n = 30–35 neurons per group from three independent cultures for (A)–(G) and six independent cultures for (H) and (I). In (B)–(D), (F), (G), and (I), * P < 0.05, ** P < 0.01, and *** P < 0.001 by one-way ANOVA followed by post-hoc Tukey’s test.

Article Snippet: The following commercially available antibodies were used for Western blot and immunostaining analysis: monoclonal mouse antibody against acetylated α-tubulin (clone 6-11B-1, Sigma), monoclonal rabbit antibody against acetylated α-tubulin (clone D20G3, Cell Signaling), monoclonal mouse antibody against GFP (clone B-2, Santa Cruz), polyclonal rabbit antibody against MAP2 (Cell Signaling), monoclonal mouse antibody against Myc (clone 9E10, Santa Cruz), monoclonal rabbit antibody against Stathmin1 (EP1573Y, Abcam), polyclonal rabbit antibody against Stathmin1 (phospho S16, Abcam), monoclonal mouse antibody against tyrosinated α-tubulin (clone TUB-1A2, Sigma), monoclonal mouse antibody against Tau (clone Tau-5, Chemicon), monoclonal mouse antibody against α-tubulin (clone DM1A, Abcam), and monoclonal mouse antibody against β3-tubulin (clone TU-20, Chemicon).

Techniques: Transfection, Staining, Expressing, Software, Phospho-proteomics

Journal: PLoS ONE

Article Title: βPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway

doi: 10.1371/journal.pone.0230814

Figure Lengend Snippet: (A) Representative images of βPix-NIKO neurons transfected with GFP and Myc, GFP-βPix-d and Myc, or GFP-βPix-d and Myc-PID at DIV3, fixed at DIV4, and stained with p-Stathmin1 (S16) (blue) and Myc antibodies (red). (B) The longest neurites shown in Fig 6A are straightened using ImageJ software. (C) The expression of GFP-βPix-d elevates the average phosphorylation levels of Stathmin1 at Ser16 in the longest neurite extending from the βPix-NIKO neurons, but the co-expression of Myc-PID with GFP-βPix-d removes the increase. (D) In the longest neurite extending from the βPix-NIKO neurons, expression of GFP-βPix-d recovers the phosphorylation levels of Stathmin1 at Ser16 but the co-expression of GFP-βPix-d and Myc-PID does not. (E) Representative images of βPix-NIKO neurons transfected with GFP and Myc, GFP-βPix-d and Myc, or GFP-βPix-d and Myc-PID at DIV3, fixed at DIV4, and stained with β3-Tubulin (blue) and Myc antibodies (red). (F) In the βPix-NIKO neurons, an increase in the longest neurite length by GFP-βPix-d is not observed when Myc-PID is co-expressed with GFP-βPix-d. (G) In the βPix-NIKO neurons, an increase in total neurite length by GFP-βPix-d is not observed when Myc-PID is co-expressed with GFP-βPix-d. (H) In the βPix-NIKO neurons, an increase in the total branching number by GFP-βPix-d is not observed when Myc-PID is co-expressed with GFP-βPix-d. n = 45–56 neurons per group from 3 independent cultures for (A)–(D) and n = 67–84 neurons per group from 3 independent cultures for (E)–(H). In (C), (D), and (F)–(H), ** P < 0.01 and *** P < 0.001 by one-way ANOVA followed by post-hoc Tukey’s test.

Article Snippet: The following commercially available antibodies were used for Western blot and immunostaining analysis: monoclonal mouse antibody against acetylated α-tubulin (clone 6-11B-1, Sigma), monoclonal rabbit antibody against acetylated α-tubulin (clone D20G3, Cell Signaling), monoclonal mouse antibody against GFP (clone B-2, Santa Cruz), polyclonal rabbit antibody against MAP2 (Cell Signaling), monoclonal mouse antibody against Myc (clone 9E10, Santa Cruz), monoclonal rabbit antibody against Stathmin1 (EP1573Y, Abcam), polyclonal rabbit antibody against Stathmin1 (phospho S16, Abcam), monoclonal mouse antibody against tyrosinated α-tubulin (clone TUB-1A2, Sigma), monoclonal mouse antibody against Tau (clone Tau-5, Chemicon), monoclonal mouse antibody against α-tubulin (clone DM1A, Abcam), and monoclonal mouse antibody against β3-tubulin (clone TU-20, Chemicon).

Techniques: Transfection, Staining, Software, Expressing, Phospho-proteomics

Journal: PLoS ONE

Article Title: βPix-d promotes tubulin acetylation and neurite outgrowth through a PAK/Stathmin1 signaling pathway

doi: 10.1371/journal.pone.0230814

Figure Lengend Snippet: βPix-d localized around microtubules activates PAK and, in turn, the active PAK phosphorylates Stathmin1 at Ser16. Phosphorylated Stathmin1 in an inactive state releases α- and β-tubulin heterodimers for microtubule polymerization and inhibits microtubule catastrophe in neurites. In summary, βPix-d promotes microtubule stabilization and neurite outgrowth.

Article Snippet: The following commercially available antibodies were used for Western blot and immunostaining analysis: monoclonal mouse antibody against acetylated α-tubulin (clone 6-11B-1, Sigma), monoclonal rabbit antibody against acetylated α-tubulin (clone D20G3, Cell Signaling), monoclonal mouse antibody against GFP (clone B-2, Santa Cruz), polyclonal rabbit antibody against MAP2 (Cell Signaling), monoclonal mouse antibody against Myc (clone 9E10, Santa Cruz), monoclonal rabbit antibody against Stathmin1 (EP1573Y, Abcam), polyclonal rabbit antibody against Stathmin1 (phospho S16, Abcam), monoclonal mouse antibody against tyrosinated α-tubulin (clone TUB-1A2, Sigma), monoclonal mouse antibody against Tau (clone Tau-5, Chemicon), monoclonal mouse antibody against α-tubulin (clone DM1A, Abcam), and monoclonal mouse antibody against β3-tubulin (clone TU-20, Chemicon).

Techniques:

Journal: Neuron

Article Title: Plug and Play Protein Modification using Homology-independent Universal Genome Engineering

doi: 10.1016/j.neuron.2019.05.047

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The IP-membrane was sequentially probed for HA-epitope (primary: Sigma H6908, 1:2000; secondary: IRDye 800CW goat anti-rabbit, 1:10,000) and the input membrane was probed for house-keeping gene GAPDH (primary: Abcam ab9485, 1:5000; secondary: IRDye 680RD goat anti-rabbit, 1:10,000).

Techniques: Recombinant, Bicinchoninic Acid Protein Assay, Clone Assay, TA Cloning, Sequencing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Filtration, Plasmid Preparation, Software, Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: The flavagline FL3 interferes with the association of Annexin A2 with the eIF4F initiation complex and transiently stimulates the translation of annexin A2 mRNA

doi: 10.3389/fcell.2023.1094941

Figure Lengend Snippet: Short-term treatment with FL3 increases the expression of AnxA2 and induces its re-localization to the PM. PC12 cells were untreated (control; Ctr), treated for 2 h with 20 nM FL3 alone or in combination with cycloheximide (CHX) or actinomycin D (Act D), as indicated (Panels A–D ). (Panel A ) IF staining was carried out using a polyclonal antibody against AnxA2 (green). The confocal images also show DNA staining (DAPI; blue) to highlight the nuclei. Scale bar: 10 µm. (Panel B ) shows the fluorescence intensity profiles of selected cells indicated by the white lines in (Panel A ) with intensity of AnxA2 shown in green and nuclear staining (DAPI) in blue. Distance is measured in pixels. (Panel C ) shows the determined areas under the peaks at the plasma membrane (PM) of the curves for AnxA2 intensity profiles shown in Panel B ( n = 3 or 4). Panel (D) 15 µg of proteins derived from the corresponding lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes for Western blot analysis with monoclonal antibodies against AnxA2. Representative blots and the results from seven independent experiments ( n = 7). (Panels E and F ): 15 µg of proteins derived from the cytoplasmic, nuclear, cytosolic (cytoplasm without mitochondria), or the mitochondrial fractions from PC12 cells without (control; Ctr) or after treatment with 20 nM FL3 were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes for Western blot analysis with monoclonal antibodies against AnxA2. Antibodies against compartment markers, i.e. the cytoplasm (tubulin; 55 kDa), nucleus (matrin 3; 125 kDa) and mitochondria (complex II; 70 kDa) were also employed as indicated. Representative blots (Panel F ) and the results from three independent experiments ( n = 3) (Panel E ) are shown. (Panel E ): The distribution of AnxA2 in the control and FL3-treated fractions as a ratio of the nucleus/cytoplasm or the mitochondria/cytoplasm AnxA2 was normalization to the loading controls (tubulin for the cytoplasmic and cytosolic fractions, matrin 3 for the nuclear fractions and complex II for the mitochondrial fractions). Statistical significance compared to control was determined by unpaired multiple t -test (* <0.05) (Panel D ) and two-way ANOVA with Tukey’s multiple comparisons test (Panel E ).

Article Snippet: Mouse monoclonal antibodies were used to detect AnxA2 (610069; BD Biosciences, Franklin Lakes, United States; dilution 1:1,000), tubulin (86298; Cell Signaling Technology, Danvers, United States; dilution 1:5,000), GAPDH (sc-32233; Santa Cruz Biotechnology, Dallas, United States; dilution 1:1,000) and complex II (459200; Invitrogen; Waltham, United States; dilution 1:1,000), whereas Matrix 3 (A300-591A, Bethyl laboratories, Montgomery, United States; dilution 1:1,000), AnxA2 (pSer25) (OAAF00618; Aviva Systems Biology, San Diego, United States; dilution 1:1,000) as well as eIF4A (C32B4) (#2013; dilution 1:1,000), p-eIF4E (#9741; dilution 1:1,000), eIF4E (#9742; dilution 1:1,000), eIF4G (#2498; dilution 1:1,000), PABP1 (#4992; dilution 1:1,000), and nucleolin (A300-711A, Bethyl Lab.; ThermoFisher Scientific, Waltham, United States; dilution 1:1,000) were detected by rabbit polyclonal antibodies all from Cell Signaling Technology (Danvers, United States).

Techniques: Expressing, Control, Staining, Fluorescence, Clinical Proteomics, Membrane, Derivative Assay, SDS Page, Western Blot, Bioprocessing

Journal: Frontiers in Cell and Developmental Biology

Article Title: The flavagline FL3 interferes with the association of Annexin A2 with the eIF4F initiation complex and transiently stimulates the translation of annexin A2 mRNA

doi: 10.3389/fcell.2023.1094941

Figure Lengend Snippet: The synthetic flavagline FL3 transiently increases the expression of AnxA2 (Panel A ) and the Ser25 phosphorylated AnxA2 form follows the level of total AnxA2 (Panel B ). Lysates were prepared from control (Ctr) PC12 cells and after their treatment for 1, 2, 6, 12, 24, and 48 h with 20 nM FL3. 10 μg of proteins separated by 10% SDS-PAGE were transferred to nitrocellulose membranes for Western blot analysis with monoclonal antibodies against AnxA2, or polyclonal antibodies against pSer25AnxA2, with tubulin serving as a loading control, as indicated. Representative blots and the results from five independent experiments ( n = 5) are shown. AnxA2 (Panel A ) is expressed in arbitrary units of intensity relative to the control sample (set = 1) after normalization to the loading control tubulin while pSer25AnxA2 (Panel B ) is expressed in arbitrary units of intensity relative to the control sample (set = 1) after normalization to total AnxA2. The standard deviations are also indicated. Statistical significance compared to control was determined by one-way ANOVA and Dunnett’s multiple comparisons test (* <0.05).

Article Snippet: Mouse monoclonal antibodies were used to detect AnxA2 (610069; BD Biosciences, Franklin Lakes, United States; dilution 1:1,000), tubulin (86298; Cell Signaling Technology, Danvers, United States; dilution 1:5,000), GAPDH (sc-32233; Santa Cruz Biotechnology, Dallas, United States; dilution 1:1,000) and complex II (459200; Invitrogen; Waltham, United States; dilution 1:1,000), whereas Matrix 3 (A300-591A, Bethyl laboratories, Montgomery, United States; dilution 1:1,000), AnxA2 (pSer25) (OAAF00618; Aviva Systems Biology, San Diego, United States; dilution 1:1,000) as well as eIF4A (C32B4) (#2013; dilution 1:1,000), p-eIF4E (#9741; dilution 1:1,000), eIF4E (#9742; dilution 1:1,000), eIF4G (#2498; dilution 1:1,000), PABP1 (#4992; dilution 1:1,000), and nucleolin (A300-711A, Bethyl Lab.; ThermoFisher Scientific, Waltham, United States; dilution 1:1,000) were detected by rabbit polyclonal antibodies all from Cell Signaling Technology (Danvers, United States).

Techniques: Expressing, Control, SDS Page, Western Blot, Bioprocessing