44-422G

antibody from Invitrogen Antibodies

Targeting: JAK1 JAK1A, JAK1B, JTK3

Provider product page for 44-422G

Western blot

Immunocytochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [21]
Comments [0]
Validations
Immunocytochemistry [2]
Other assay [9]

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Product number: 44-422G - Provider product page
Provider: Invitrogen Antibodies
Product name: Phospho-JAK1 (Tyr1034, Tyr1035) Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This product was formerly called "Phospho-JAK1 (Tyr1022, Tyr1023) Polyclonal Antibody" because Human Jak1 residues Tyr1034 and Tyr1035 have been historically referenced as Tyr1022 and Tyr1023.
Reactivity: Human, Mouse
Host: Rabbit
Isotype: IgG
Vial size: 100 μL
Storage: -20°C

JAK1 protein structure - 44-422G shown in red.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of JAK1 (pY1022/1023) was done on 70% confluent log phase COLO 205 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with JAK1 (pY1022/1023) Rabbit polyclonal Antibody (Product # 44-422G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing membrane localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of JAK1 (pY1022/1023) was done on 70% confluent log phase COLO 205 cells. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with JAK1 (pY1022/1023) Rabbit polyclonal Antibody (Product # 44-422G) at 1:250 dilution in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Fluor 488 Goat Anti-Rabbit IgG Secondary Antibody (Product # A-11008) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor 594 Phalloidin (Product # A12381). Panel d is a merged image showing membrane localization. Panel e shows no primary antibody control. The images were captured at 20X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 6 Transformed LTK-F568L BaF3 cells are sensitive to the ALK inhibitor PF-2341066. ( A ) BaF3 cells transformed to cytokine independence by LTK-F568L were left untreated (0.1% DMSO, black circles) or cultured in the presence of 0.5 uM (blue squares), 1 uM (green triangles), or 2 uM (red inverted triangles) of the cMET/ALK inhibitor PF2341066 (PF) on Day 0. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. ( B ) IL-3-dependent (+IL-3) BaF3 cells harboring ALK-F1174L were cultured without (black circles) or with (blue squares) 0.5 uM PF2341066. BaF3 cells transformed to IL-3-independence by ALK-F1174L were cultured without (green triangles) or with (red inverted triangles) 0.5 uM PF2341066. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. ( C ) Parental BaF3 cells were cultured without (black circles) or with (blue squares) 0.5 uM PF2341066. BaF3 cells expressing wildtype LTK were cultured without (green triangles) or with (red inverted triangles) 0.5 uM PF2341066. The total number of viable cells for each treatment was determined daily by trypan blue exclusion. ( D ) IL-3-dependent BaF3 cells expressing LTK-F568L were cultured without (black inverted triangles) or with (blue circles) 0.5 uM PF2341066. BaF3 cells transformed to IL-3 independence by LTK-F568L were cultured without (green squares) or with (red diamonds) 0.5 uM PF2341066. The total number of viable cells for ea

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 2 Expression of LTK constructs in 293T cells. ( A ) Relative protein expression of wildtype and mutant LTK constructs in 293T cells. Lysates of 293T cells transiently expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were immunoblotted with an anti-HA antibody as well as HSP90 as a loading control. ( B ) 293T cell lysates containing the LTK proteins were treated with PNGase F (lanes 3, 5, and 7) or left untreated (lanes 2, 4, and 6) and immunoblotted with HA antibodies and HSP90 as a loading control. ( C ) HA-tagged LTK proteins expressed in 293T cells were immunoprecipitated and immunoblotted with HA antibodies as well as anti-phosphotyrosine (pTyr) antibodies (top). Antibodies that recognize the indicated proteins (Shc, pShC-Y239/Y240, ERK, pERK-T202/Y204, JAK1, pJAK1-Y1022/Y1023, JAK2, pJAK2-Y1007/Y1008, AKT, and pAKT-S473) were used to immunoblot cell lysates of 293T cells expressing LTK proteins (bottom). The phosphorylated form of the protein is designated with a ""p"" preceding the protein name.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 3 LTK-F568L transforms BaF3 cells to cytokine-independence. ( A ) BaF3 cells stably expressing LTK-F568L (red inverted triangles), LTK-R669Q (green triangles), or wildtype LTK (blue squares) and parental control cells (black circles) were cultured in the absence of IL-3 at Day 0. The total number of viable cells was determined at each timepoint by trypan blue exclusion. Similar results were obtained in three independent experiments. The dashed line indicates cell numbers going below the detection of the hemacytometer towards zero. ( B ) Lysates from cells stably expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were cultured in the absence of IL-3 for six hours and immunoprecipitated with HA antibodies, followed by immunoblotting with HA and pTyr antibodies (first two blots). The same lysates were immunoblotted with antibodies to the indicated cell signaling proteins (see Figure 2 legend plus pShc-Y317, STAT3, pSTAT3-Y705, STAT5 and pSTAT5-Y694). Cell lysate from IL-3-independent LTK-F568L-expressing BaF3 cells was also analyzed (lane 5).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 4 LTK-F568L transforms 32D cells to cytokine-independence. ( A ) 32D cells stably expressing LTK-F568L (red inverted triangles), LTK-R669Q (green triangles), or wildtype LTK (blue squares) and parental control cells (black circles) were cultured in the absence of IL-3 at Day 0. The total number of viable cells was determined at each timepoint by trypan blue exclusion. Similar results were obtained in three independent experiments. The dashed line indicates cell numbers going below the detection of the hemacytometer towards zero. ( B ) Lysates from cells stably expressing either HA-tagged vector control (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were cultured in the absence of IL-3 for six hours and immunoprecipitated with HA antibodies, followed by immunoblotting with HA and pTyr antibodies (first two blots). The same lysates were immunoblotted with antibodies to the indicated cell signaling proteins (See Figure 3 legend). Cell lysate from IL-3-independent LTK-F568L-expressing 32D cells was also analyzed (lane 5).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 5 Transformation of BaF3 cells by LTK-F568L requires JAK family kinase activity. ( A ) BaF3 cells transformed to cytokine independence by LTK-F568L were cultured in the presence of 0.5 uM (green squares) or 1 uM (blue triangles) JAK Inhibitor I (JI) or 0.1% DMSO (red circles) on Day 0. The total number of viable cells was determined by trypan blue exclusion. Dashed lines indicate viable cell counts going below the limit of detection of the hemacytometer towards zero. Similar results were obtained in two independent experiments. ( B ) IL-3-independent LTK-F568L BaF3 cells were treated with 1 uM (lane 2) JI or DMSO (lane 1) for three hours and immunoblotted for the indicated proteins (See Figure 3 legend). Phosphotyrosine (pTyr) on LTK-F568L was detected by immunoblotting following immunoprecipitating LTK-F568L with HA antibodies.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Figure 7 LTK-F568L and LTK-R669Q transform epithelial cells. ( A ) Cell lysates from RIE cells stably expressing either empty vector (v, lane 1), wildtype LTK (wt, lane 2), LTK-R669Q (RQ, lane 3), or LTK-F568L (FL, lane 4) were analyzed by immunoprecipitation and immunoblotting, as indicated (see Figure 3 legend). ( B ) RIE cells stably expressing either wildtype LTK (wt), LTK-F568L (F568L), or LTK-R669Q (R669Q) were plated and reached confluency after six days. Cells were cultured further and growth was grossly assessed for the loss of contact inhibition. Cells expressing wildtype LTK did not grow beyond confluency, continuing to exhibit contact inhibition. However, cells expressing LTK-F568L continued to proliferate, forming swirling patterns of cells growing on top of the monolayer. Cells expressing LTK-R669Q formed distinct secondary morphological structures as these cells formed compact clusters of cells that grew on top of the monolayer of cells. Representative photographs of cells are shown: plates were stained with crystal violet (top row) after photographs were taken at 10x with a digital camera (middle) and 50x with a Zeiss Automated Fluorescent Microscope using AxioVision (bottom row). Results shown are representative of similar findings of three independent experiments.