gal4 activation domain vector pact2  (TaKaRa)

Journal: Journal of Cell Science

Article Title: Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

doi: 10.1242/jcs.170639

Figure Lengend Snippet: The position of Ball in the IFM and the effect of reduced expression. (A) Ball in the IFM. Myofibrils were labelled with anti-Ball antibody (green) and with anti-Kettin antibody as a Z-disc (Z) marker (red). In wild-type (wt) myofibrils, Ball is labelled at the Z-disc and, more weakly, across the sarcomere. No Ball labelling is detected in myofibrils of Ball-knockdown flies. (B) Abnormal sarcomeres in IFM of Ball-knockdown flies compared with sarcomeres in wild-type IFM. Myofibrils were labelled for actin with phalloidin (red), at the M-line (M) with anti-obscurin antibody (green) and at the Z-disc with anti-Kettin antibody (magenta). In Ball-knockdown flies, labelling of actin, the M-line and the Z-disc is less regular than in the wild-type. Obscurin is present in the M-line, but labelling is narrower than in the wild-type; Z-discs in this example appear fuzzy at the periphery (arrows). Scale bars: 5 µm. (C) Flight tests of wild-type and Ball-knockdown flies. The percentage of flies able to fly is shown, n=20 for each genotype. Flies were flightless in Mef2-GAL4;UAS-ball1(ex2)-IR and Mef2-GAL4;UAS-ball2(ex1-2)-IR RNAi lines. Control flies with UAS-ball-IR or Mef2-GAL4 alone flew normally. Crosses with UAS-ball1(ex2)-IR were performed at 29°C and UAS-ball2(ex1-2)-IR at 25°C. (D) Electron microscopy images of wild-type and Ball-knockdown flies. Z-discs of IFM in Ball-knockdown flies are often fragmented and shifted towards the core of the myofibril (arrows), leading to fraying of thick and thin filaments, and M-line disruption at the periphery. In several cases, Z-discs are broken and do not span the entire myofibril, with thick and thin filaments bypassing the Z-disc (asterisks). Scale bar: 500 nm.

Article Snippet: The strains containing the bait constructs were subsequently co-transformed with a Drosophila whole-adult fly cDNA library cloned in the pACT2 - Gal4 activation domain vector (Clontech).

Techniques: Expressing, Marker, Electron Microscopy

Journal: Journal of Cell Science

Article Title: Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

doi: 10.1242/jcs.170639

Figure Lengend Snippet: The effect of reduced obscurin expression on Ball and MASK. (A) IFM myofibrils were labelled with phalloidin (red), anti-Ball (green) and anti-Kettin (magenta) antibodies. Wild-type (wt) myofibrils show clear labelling at the Z-disc (Z); Ball was also detected at the M-line (M), in addition to some labelling across the sarcomere. In obscurin-knockdown flies (Mef2-GAL4;UAS-unc89-IR), Ball is in the Z-disc, but labelling is more spread out than in the wild-type. Ball is diffusely distributed across the sarcomere and there is no clear M-line labelling. Arrows mark the M-line. (B) IFM myofibrils were labelled with phalloidin and anti-Kettin as for A, and anti-MASK (green). In obscurin-knockdown flies, MASK is present in the Z-discs, but absent or spotty in the M-lines (arrows). The brightness of the Mef2-GAL4;UAS-unc89-IR MASK labelling has been slightly increased in order to see remnants of MASK in the M-line of the merged image. Scale bars: 5 µm.

Article Snippet: The strains containing the bait constructs were subsequently co-transformed with a Drosophila whole-adult fly cDNA library cloned in the pACT2 - Gal4 activation domain vector (Clontech).

Techniques: Expressing

Journal: Journal of Cell Science

Article Title: Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle

doi: 10.1242/jcs.170639

Figure Lengend Snippet: The position of MASK in the IFM and the effect of reduced expression. (A) MASK in the IFM. Myofibrils were labelled with phalloidin (red), and anti-MASK (green) and anti-Kettin (magenta) antibodies. In wild-type (wt) myofibrils, MASK is in the M-line (M) and Z-disc (Z), with the Z-disc labelled more strongly by the antibody than the M-line. MASK is greatly reduced in MASK-knockdown flies. The brightness of the Mef2-GAL4;UAS-MASK(ex1)-IR Kettin labelling has been slightly increased in order to see Kettin clearly in the merged image. (B) Abnormal sarcomeres in IFM of MASK-knockdown flies compared with wild-type IFM. Myofibrils were labelled with phalloidin, and anti-Kettin antibodies as in A and with anti-obscurin antibody (green). In MASK-knockdown flies, M-lines and Z-discs are evenly spaced, but sarcomeres are shorter. M-lines and corresponding H-zones are often bent (arrows). MASK-knockdown flies were eclosed ‘escapers’. Scale bar: 5 µm. (C) Flight tests of wild-type and MASK-knockdown flies. The percentage of flies able to fly is shown, n=20 for each genotype. Flies were flightless in Mef2-GAL4;UAS-MASK(ex1)-IR and Mef2-GAL4,UAS-MASK(ex6)-IR RNAi lines. Control flies with UAS-MASK-IR or Mef2-GAL4 alone flew normally. Crosses with UAS-mask(ex1)-IR flies were at 29°C, and UAS-mask(ex6)-IR at 25°C.

Article Snippet: The strains containing the bait constructs were subsequently co-transformed with a Drosophila whole-adult fly cDNA library cloned in the pACT2 - Gal4 activation domain vector (Clontech).

Techniques: Expressing