Anti-alpha Tubulin antibody - Loading Control

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB4074)

ab4074 staining alpha Tubulin in HeLa cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab4064 at 1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• WB

Project

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

ab4074 cross-reacts strongly with Mouse 3T3 lysate and shows weak cross-reactivity with Rat liver, Cow, Chicken and CHO lysates.

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab4074) at 1 µg/mL

Lane 1:

Mouse 3T3 lysate at 20 µg

Lane 2:

Rat liver lysate at 20 µg

Lane 3:

Cow cell lysate at 20 µg

Lane 4:

Chicken cell lysate at 20 µg

Lane 5:

CHO lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/ko/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab6721'>ab6721</a>) at 1/5000 dilution

Predicted band size: 50 kDa

Observed band size: 50 kDa

true

Exposure time: 30s

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Immunocytochemistry of SisNPFR-C6E8 cell line. Cells were prefixed in freshly prepared 4% paraformaldehyde (PFA) in F12 K culture medium with 10% fetal bovine serum and Geneticin. Fixed cells were permeabilized in PBST (0.25% Triton X-100 in PBS) for 5 min and blocked at 4°C overnight in 5% normal goat serum. The cells were then incubated at 4°C overnight with ab4074 (1 : 50). Cover slips were placed onto slides and mounted with VectaShield containing DAPI.

Image from PLoS One. 2014; 9(10): e109590. Fig 5o,doi: 10.1371/journal.pone.0109590 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

Ap

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab4074) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 20 µg

Lane 2:

HEK-293 (Human) Whole Cell Lysate (ab52256) at 20 µg

Lane 3:

Western blot - NIH/3T3 whole cell lysate (<a href='/ko/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 20 µg

Lane 4:

PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 20 µg

Lane 5:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 20 µg with Human alpha Tubulin peptide (<a href='/ko/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Lane 6:

HEK-293 (Human) Whole Cell Lysate (ab52256) at 20 µg with Human alpha Tubulin peptide (<a href='/ko/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Lane 7:

Western blot - NIH/3T3 whole cell lysate (<a href='/ko/products/cell-lysates/nih-3t3-whole-cell-lysate-ab7179'>ab7179</a>) at 20 µg with Human alpha Tubulin peptide (<a href='/ko/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Lane 8:

PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 20 µg with Human alpha Tubulin peptide (<a href='/ko/products/unavailable/human-alpha-tubulin-peptide-ab23537'>ab23537</a>)

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Predicted band size: 50 kDa

false

• WB

Ap

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

All lanes:

Western blot - Anti-alpha Tubulin antibody - Loading Control (ab4074) at 1 µg/mL

Lane 1:

HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate (ab27252) at 10 µg

Lane 2:

NIH/3T3 (Mouse) Whole Cell Lysate (ab52956) at 10 µg

Lane 3:

PC-12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate (ab50957) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) (ab65484) at 1/3000 dilution

Predicted band size: 50 kDa

false

• WB

CiteAb

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Western Blotting using Anti-alpha Tubulin antibody - Loading Control, ab4074. Publication image from Siekierska, A. et al., 2019, Nat Commun, 30755616. Legend direct from paper.

In vitro studies support variant pathogenicity. a Western blot performed on patient-derived fibroblasts from patients 4 and 5 of family 3 carrying the L78Rfs*35/R942Q VARS variant showed almost 50% reduction in VARS protein. Values are mean of three separate experiments. Error bars represent SD. b RT-qPCR on the fibroblasts showed almost complete absence of the frameshift allele at mRNA level, treatment with cycloheximide caused a partial increase in expression of the frameshift allele, which was not seen with DMSO-treated control. Values are mean of three separate experiments performed in triplicate. Error bars represent SD. c Immunocytochemistry highlighted the nucleus (Hoechst), VARS and KDEL, a marker for endoplasmatic reticulum. VARS co-localizes with KDEL. There was no difference in localization between the control line and the patient fibroblasts. d VARS and TARS aminoacylation activity measured in extracts from the patient fibroblasts. The data were normalized to ATTC fibroblasts. VARS aminoacylation activity was measured in technical triplicate at three separate passages, and TARS activity was measured once in technical triplicate. Data are represented as mean-specific activity and error bars represent SEM. * indicates significant difference from control. e A haploid yeast strain deleted for endogenous VAS1 was transformed with a LEU2-bearing pRS315 vector containing wild-type VAS1, the indicated mutant form of VAS1, or no insert (empty). Cultures for each strain (labeled along the top) were either undiluted (UD) or diluted 1 : 10 or 1 : 100 and then spotted on solid medium containing 5-FOA to determine whether the VAS1 alleles complement loss of endogenous VAS1 at 30 °C. Only G822S shows absent growth indicating a functional null allele. f VARS and TARS aminoacylation activity measured in extracts from patient-derived lymphoblasts of patients 1 and 2 (L434V/G822S) and their parents and patient 9 (R404W). Data were normalized to the paternal cells. VARS aminoacylation activity was measured in technical triplicate at three separate passages, and TARS activity was measured once in technical triplicate. Data are represented as mean-specific activity and error bars represent SEM. * indicates significant difference from L434V paternal lymphoid cells. In a, b, d, and f one-way ANOVA with Tukey’s multiple comparisons test was used. Significant values are noted **p < 0.01, ***p < 0.001, and ****p < 0.0001

false

• WB

CiteAb

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Western Blotting using Anti-alpha Tubulin antibody - Loading Control, ab4074. Publication image from Zhang, L. et al., 2018, Nat Commun, 30575721. Legend direct from paper.

PHD2 expression and function in human melanocytes and melanomas. a IHC staining of PHD2 in 126 nevus and 266 melanoma specimens. A melanoma progression tissue microarray (TMA) was stained with the anti-PHD2 antibody and PHD2 staining positive and negative cases were calculated. Representative PHD2 positive (upper panel) and negative cases (lower panel) are shown. Bars indicate 100 µm. b Percentage of cases with PHD2 expression. There was significantly less percentage of melanoma cases with PHD2 expression than that of nevi; *p < 0.01. c. TCGA SKCM patients were sorted into PHD2-high and PHD2-low groups according to PHD2 mRNA expression levels (top 5% versus bottom 5%). dPHD2 expression and survival curve. The Kaplan–Meier survival curves showed that the PHD2-high group have a significantly improved patient survival than PHD2-low group (log rank p value = 0.00643). ePHD2 knockdown induces Akt phosphorylation. Immortalized human melanocytes (hTERT/p53DD/Cdc24R24C-BRAFV600E) were transfected with two independent PHD2 shRNAs. Cells transfected with shGFP were included as a negative control. Western blots were performed using the indicated antibodies. f Reintroduction of PHD2 in PHD2−/− MEFs reduces Akt phosphorylation. PHD2−/− MEFs (left lane) were transfected with wild-type PHD2 (lane adjacent to the left lane). PHD2-positive MEFs which express endogenous PHD2 were used as a control (right lane). g DFO or DMOG treatment. Immortalized human melanocytes were transfected with control vectors or EGLN knockdown vectors. These cells were than treated with DFO or DMOG. h Overexpressed HIF-2α in immortalized human melanocytes. i The melanoma-derived PHD2-P317 mutation inhibits PHD2 binding to HIF-1α. Biotin-labeled HIF1α-ODD peptides were incubated with WCLs derived from HEK293T cells transfected with the indicated PHD2 constructs (beads as negative control). Binding of PHD2 to HIF1α-ODD is abolished with the P317 mutation. j The PHD2-P317 mutation inhibits PHD2 and HIF-1α interaction. Unlike WT-PHD2, P317S-PHD2 is largely impaired in inhibiting HRE reporter activities detected by the Dual-Luciferase Reporter Assay System. The error bars indicate s.d.

false

• WB

CiteAb

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Western Blotting using Anti-alpha Tubulin antibody - Loading Control, ab4074. Publication image from Shi, M. et al., 2018, Nat Commun, 30478271. Legend direct from paper.

Signaling components essential for the AA-stimulated retrograde trafficking. All cells are HeLa cells. a Cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 µM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. b Endogenous SLC38A9 was depleted by lentivirus-transduced shRNAs as assessed by RT-qPCR from n = 3 independent experiments. c The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. Knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for phospho-S6K1 (p-S6K1) and GAPDH. d SLC38A9 is required for the AA-simulated Golgi trafficking. Cells treated with indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a. e Immuno-blots showing that endogenous Lamtor1 and RagA/B were depleted by respective lentivirus-transduced shRNAs. f Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was similar to d. g, h Under Lamtor1 knockdown condition similar to e, an RNAi-resistant Lamtor1 was able to express and rescue the AA-stimulated Golgi trafficking. i Lamtor1 is required for the AA-stimulated reduction of surface CD8a-furin-mEos2. Knockdown and surface labeling were similar to e and Fig. 2j, respectively. Surface intensity was normalized by mEos2 total intensity. Surface DMEM/HBSS-ratio is the normalized surface intensity under DMEM divided by that under HBSS treatment. j mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin, or 250 nM Torin1 was present throughout the treatment. In a, b, d, f, h–j, the displayed value is the mean of n = 3 independent experiments and individual data points are shown as red dots. Error bar, mean ± s.d.; P values were from t test (unpaired and two-tailed); N.S., not significant (P > 0.05); *P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA

false

• WB

CiteAb

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Western Blotting using Anti-alpha Tubulin antibody - Loading Control, ab4074. Publication image from Shi, M. et al., 2018, Nat Commun, 30478271. Legend direct from paper.

Signaling components essential for the AA-stimulated retrograde trafficking. All cells are HeLa cells. a Cells stably expressing CD8a-furin were starved in HBSS for 2 h followed by surface-labeling and subsequent incubation with either HBSS or DMEM for 20 min. 1% DMSO or 2.5 µM conA was present throughout the incubation. Cells were stained and the AA-stimulated Golgi trafficking is quantified by imaging. b Endogenous SLC38A9 was depleted by lentivirus-transduced shRNAs as assessed by RT-qPCR from n = 3 independent experiments. c The knockdown of endogenous SLC38A9 attenuated the AA-stimulated mTORC1 activity. Knockdown cells were incubated with DMEM/-AAs for 50 min followed by incubation with DMEM for 20 min. Cell lysates were immuno-blotted for phospho-S6K1 (p-S6K1) and GAPDH. d SLC38A9 is required for the AA-simulated Golgi trafficking. Cells treated with indicated shRNAs were transfected to express CD8a-furin and subjected to treatment and analysis similar to a. e Immuno-blots showing that endogenous Lamtor1 and RagA/B were depleted by respective lentivirus-transduced shRNAs. f Lamtor1 and 3 but not RagA/B are required for the AA-stimulated Golgi trafficking. The experiment was similar to d. g, h Under Lamtor1 knockdown condition similar to e, an RNAi-resistant Lamtor1 was able to express and rescue the AA-stimulated Golgi trafficking. i Lamtor1 is required for the AA-stimulated reduction of surface CD8a-furin-mEos2. Knockdown and surface labeling were similar to e and Fig. 2j, respectively. Surface intensity was normalized by mEos2 total intensity. Surface DMEM/HBSS-ratio is the normalized surface intensity under DMEM divided by that under HBSS treatment. j mTORC1 is not required for the AA-stimulated retrograde trafficking. The experiment was conducted similarly to a except that 1% DMSO, 100 nM rapamycin, or 250 nM Torin1 was present throughout the treatment. In a, b, d, f, h–j, the displayed value is the mean of n = 3 independent experiments and individual data points are shown as red dots. Error bar, mean ± s.d.; P values were from t test (unpaired and two-tailed); N.S., not significant (P > 0.05); *P ≤ 0.05. GL2 is a non-targeting control siRNA or shRNA

false

• WB

CiteAb

Western blot - Anti-alpha Tubulin antibody - Loading Control (AB4074)

Western Blotting using Anti-alpha Tubulin antibody - Loading Control, ab4074. Publication image from Moen, A. et al., 2016, Nat Commun, 27321414. Legend direct from paper.

Methylation status of HSPA1-K561 in subcellular fractions.Top panel, methylation status in whole-cell extracts (WCE) as well as in cytosolic (Cyt) and nuclear (Nuc) fractions from HeLa and HEK-293 cells was determined by mass spectrometry as in Fig. 1a. Bottom panel, western blot analysis of total HSPA1, alpha-tubulin (cytosolic marker) and histone H3 (nuclear marker) in protein samples analysed in the upper panel.

false